qPCR Technical Guide

Detection Methods

Primer and Probe Design

Instrumentation

Applications Guide qPCR Technical Guide qPCR ApplicationsGuide. Instrumentation. Primer andProbe Design. qPCR DetectionMethods. Quantitative PCR:Howdoesitwork?. Introduction. Table ofContents Level ofQuantitativePCRControls . Quality oftheTemplate. Low-Throughput qPCRSystems . High-Throughput qPCRInstruments. Considerations. Dye ChoiceandQuenchers. Template, PrimerDesign,Probe Design, Modifications. Structured Probes. Linear Probes. Probe-Based Detection. Dye-Based Detection. umr...... 17 Summary...... Qualitative Analysis. Relative Quantitation. Absolute Quantitation. Roche AppliedScience. Corbett Research. Cepheid. Bio-Rad Laboratories. Techne. Stratagene. Roche AppliedScience. Applied Biosystems(ABI). Software. Detection Linearity. Dynamic Range. Specificity ofDetection. Sensitivity. Components. Primer andProbe DesignSoftware andWeb Sites. Quenchers inProbe Design. Dye ChoiceinProbe Design. Probe DesignConsiderations. Primer DesignConsiderations. Template Considerations. Minor Groove Binders. Locked NucleicAcid. Scorpions Probes. Molecular BeaconsProbes. Hybridization Probes. (Dual LabeledFluorescent Probes orTaqMan). Hydrolysis Probes Advantages andDisadvantagesofDye-BasedDetection. Melt/Dissociation Curves. DNA BindingDyes–HowTheyWork. sigma.com ...... 14 ...... 16 ...... 1 ...... 12 ...... 12 ...... 14 ...... 8 ...... 6 ...... 12 ...... 12 ...... 12 ...... 7 ...... 12 ...... 16 ...... 7 ...... 3 ...... 12 ...... 17 ...... 8 ...... 6 ...... 5 ...... ORDER: 800-325-3010 ...... 17 ...... 17 ...... 15 ...... 17 ...... 17 ...... 9 ...... 6 ...... 3 ...... 16 ...... 13 ...... 12 ...... 13 ...... 9 ...... 9 ...... 3 ...... 7 ...... 10 ...... 10 ...... 16 ...... 10 ...... 9 ...... 17 ...... 13 ...... 2 ...... 3 ...... TECHNICAL SERVICE: 800-325-5832 ...... 6 ...... 11 ...... 4 ..... Troubleshooting. qPCR ReagentSelectionTable.

Quantitative Real-TimeRT-PCR. Appendix 2:HowtoOptimizeYour Appendix 1:Traits ofCommonlyUsedFluorophores .

References. Optimizing qPCR.

Fluorescence Issues. Dissociation/Melting Curves. Reverse Transcription. RNA QuantityandIntegrity. SamplingandRNAExtraction. Tissue Introduction. Product Specific. Multiplex. qRT-PCR Specific. Standard Curve. Elevated Fluorescence Acquisition. Crossing PointDataEvaluation. Guidelines forOptimizingBothqPCRandqRT-PCR . Additional OptimizationforMultiplexReactions. Transcription PCR(qRT-PCR). Additional GuidelinesforQuantitativeReverse Check PrimerDesignforPrimer-Dimer Potential. Optimize Mg Optimize PrimerConcentrations. Check PrimerDesign. Optimize ReverseTranscription. Conduct No-ReverseTranscriptase Controls (no-RT) . Confirm thatPrimersSpanorFlankLongIntrons. Verify RNAQuality. Set theThreshold Value. Prepare aMeltCurve. Validate PerformancewithaStandard Curve. Optimize Probe Concentration. Optimize PrimerConcentrations...... 33 ...... 38 ...... 35 ...... 2+ ...... 29 ...... 31 ...... 33 ...... 18 ...... 33 ...... Cnetain...... 26 Concentration...... 29 ...... 36 ...... 22 ...... 26 ...... 21 ...... 21 ...... 35 ...... 30 ...... 22 ...... 28 ...... 37 ...... 35 ...... 24 ...... 19 ...... 36 ...... 26 ...... 18 ...... 35 ...... 20 ...... 26 ...... 18 ...... 18 ...... 22 ...... 23 ...... 34 ......

INNOVATION @ WORK

Introduction

The routine study of DNA became practical with the Numerous qPCR detection chemistries and instruments invention of the polymerase chain reaction (PCR) by are now available to answer a wide range of questions. Kary Mullis in 1983. With the advent of PCR, it was For instance, qPCR can be used to measure viral load or possible to multiply a given DNA segment from com- bacterial pathogens in a clinical sample, to verify qPCR Technical Guide plex genetic material millions of times in a few hours microarray data, for allelic discrimination or to deter- using simple equipment. PCR provided researchers with mine RNA (via cDNA) copy numbers to analyze the level the ability to generate enough genetic material to study of gene product in a tissue sample. gene function and the effects of mutations, offering The Quantitative PCR Technical Guide from Sigma-Aldrich new possibilities in basic research and diagnostics. is intended to provide new users with an introduction Despite these advances, quantitation of DNA or RNA in to qPCR, an understanding of available chemistries, and cells remained a difficult task until 1993 when Russell the ability to apply qPCR to answer research questions. Higuchi, et al.,1 introduced real-time, or kinetic, moni- The guide also contains numerous tips and tools for the toring of DNA amplification. Higuchi’s experiments experienced qPCR user. revealed that the relationship between the amount of target DNA and the amount of PCR product generated after a specific number of amplification cycles is linear. This observation formed the basis for real-time quanti- tative PCR (qPCR).

Our Innovation, Your Research — Shaping the Future of Life Science 1 2 Quantitative PCR: How does it work? the fluorescence plot,andthelowerCtvalue. sample DNA,thesooneraccumulatedproduct isdetectedin number ofspecificamplicons.Thehighertheinitialamount threshold level,itisassumedthatallreactions contain anequal reactions atanygivenfluorescence level.Therefore, atthe In theory, anequalnumberofmoleculesare present inallofthe a threshold 10-foldhigherthanthisaverage. determining thebaseline(background) averagesignalandsetting cally calculatethethreshold leveloffluorescence signal by (which lookslinearinthelogphase).Mostinstrumentsautomati- amplification baselineandwithintheexponentialincrease phase the initialcopynumber. Thethreshold shouldbesetabovethe DNA inthesamples.Itisinverselycorrelated tothelogarithmof This threshold mustbeestablishedtoquantifytheamountof The CtValue isthemostimportantparameterforquantitativePCR. cycle orCtValue. significant above the baseline or background, is called the thresholdThe pointatwhichfluorescence isfirstdetectedasstatistically the amountoftemplatepresent atthebeginningofreaction. between PCR product and fluorescence intensity is used to calculateDNA concentrationoverabroad range,andthelinearcorrelation cation process. Thefluorescence signalisdirectly proportional to fluorescence valuesare recorded duringeachcycleoftheamplifi- hybridizing probe, totheaccumulatingDNAmolecules,and a fluorescent dyebinds,eitherdirectly orindirectly viaalabeled as theyare produced usingafluorescent label.Duringamplification, The concept of qPCR is simple: amplification products are measured only attheendpointofamplification. with traditionalPCR,theamountofPCRproduct ismeasured PCR product ismeasured aftereachround ofamplificationwhile PCR. Themajordifference beingthatwithqPCRtheamountof quantitativePCR(qPCR)isverysimilartotraditional Real Time Quantitative PCR:Howdoesitwork?

Figure 1.PCRAmplificationPlots Relative Fluorescence 0.25 0.75 sigma.com 0.5 0 1 Baseline 10 Standard X-Y Plot X-Y Standard ORDER: 800-325-3010 Cycle Number Linear 20 Exponential 30 TECHNICAL SERVICE: 800-325-5832 Plateau 40

fluorescent signal. DNA concentrationbyextrapolatingbackfrom areliable detection. Bothmethodsrely oncalculatingtheinitial(zero cycle) dye-based, ornon-specificdetection,andprobe-based, orspecific There are twomainmethodsusedtoperformquantitative PCR: yield, ortheplateauphase. in thelinearplot.Afterthispoint,reaction isatthemaximum reagent, thereaction reaches alinearphase,whichisbesttracked Once thereaction reaches significantproduct inhibition,orlimiting exponential phase,whichisbesttrackedinthesemi-logplot. line, thereaction coursecanbefollowedreliably through the yield hasreached thedetectionthreshold, shownasthedotted The background signalisshownasbaselineinFigure 1.Afterthe the resulting product-related fluorescence istoolowtobedetected. the initialconcentrationoftemplateisextremely low;therefore size different reaction phases.DuringatypicalqPCRexperiment, of PCRamplification.Thedifferent graphingtechniquesempha- Both plotscanbebroken intodifferent regions showingthephases can beusedtocalculatetheefficiencyofPCR. region ofthegraph.Theslopethisportionsemi-logplot geometric reaction willresult inastraightlinetheexponential does inthestandard X-Yplot.Alogarithmicplotofasuccessful of thedatashouldshowaclassicexponentialamplificationasit geometric amplification,ideallydoublingeverycycle,alinearplot of logfluorescence versuscylenumber(Fig.1).SincePCRisa X-Y plotoffluorescence versuscyclenumberandasemi-logplot There are twowaystographqPCRfluorescence data:astandard

Log (Relative Fluorescence) –3 –2 –1 0 Baseline 10 Semi-log Plot Semi-log Cycle Number Exponential Linear 20 Cycle threshold Ct ≅15.5 30 Plateau 40

INNOVATION @ WORK qPCR Detection Methods

Both dye-based and probe-based qPCR detection methods utilize binding dye SYBR® Gold, to DNA. A selection of commonly used a fluorescent signal to measure the amount of DNA in a sample. DNA binding dyes is presented in Table 1. Fluorescence results from the molecular absorption of light energy Table 1. DNA Binding Dyes

by fluorescent compounds, fluorophores, at one wavelength and qPCR Detection Methods the nearly instantaneous re-emission at another, longer wavelength DNA Binding Dyes of lower energy. The fluorescence signature of each individual SYBR® Green I fluorophore is unique in that it provides the wavelengths and ® amount of light absorbed and emitted. During fluorescence, the SYBR Gold absorption of light excites electrons to a higher electronic state YoYo™-1 –8 where they remain for about 1-10 × 10 seconds and then they Yo-Pro™-1 return to the ground state by emitting a photon of energy. The BOXTO intensity of the emitted light is a measure of the number of photons emitted per second. BEBO ™ Most fluorophores are either heterolytic or polyaromatic hydro- Amplifluor carbons. Fluorescence intensity depends on the efficiency with Quencher-labeled primers I which fluorophores absorb and emit photons, and their ability to Quencher-labeled primers II undergo repeated excitation/emission cycles. The maximal absorption ™ and emission wavelengths, and the excitation coefficients, of the LUX primers most common fluorophores are listed in Appendix 1: Traits of Prior to binding DNA, these dyes exhibit low fluorescence. During Common Fluorophores. amplification, increasing amounts of dye bind to the double- Figure 2 below shows the emission curves of a selection of stranded DNA products as they are generated. For SYBR Green I, common fluorophores. after excitation at 497 nm (SYBR Gold 495 nm), an increase in emission fluorescence at 520 nm (SYBR Gold 537 nm) results during the polymerization step followed by a decrease as DNA is Dye-Based Detection denatured. Fluorescence measurements are taken at the end of the elongation step of each PCR cycle to allow measurement of Dye-based detection is performed via incorporation of a DNA DNA in each cycle. See Figure 3, for an illustration of how a dye binding dye in the PCR. The dyes are non-specific and bind to any based assay works. Assays using SYBR Green I binding dye are less double-stranded DNA (dsDNA) generated during amplification specific than conventional PCR with gel detection because the resulting in the emission of enhanced fluorescence. This allows specificity of the reaction is determined entirely by the primers. the initial DNA concentration to be determined with reference However, additional specificity can be achieved and the PCR can to a standard sample. be verified by melt or dissociation curves. DNA Binding Dyes – How They Work Melt/Dissociation Curves DNA binding dyes bind reversibly, but tightly, to DNA by intercalation, Melt curves allow a comparison of the melting temperatures of minor groove binding, or a combination of both. Most real-time amplification products. Different dsDNA molecules melt at PCR assays that use DNA binding dyes detect the binding of the different temperatures, dependent upon a number of factors fluorescent binding dye SYBR® Green I, or the more stable including GC content, amplicon length, secondary and tertiary

Figure 2. Fluorescent Emission of Common Fluorophores Emission Curves 1000 900 800 700 600 500 400 300 Fluorescence (au) 200 100 0 100 390 410 430 450 470 490 510 530 550 570 590 610 630 650 670 690 710

Wavelength (nm)

■ Alexa dye (346) ■ FAM (420) ■ TET ■ JOE (500) ■ Cy3 (520) ■ Cy3 (528) ■ REX (520) ■ Tamra ■ RR (540) ■ ROX dye (550) ■ BTR (550) ■ TR (550) ■ Cy5

Our Innovation, Your Research — Shaping the Future of Life Science 3 4 qPCR Detection Methods n Advantages Dye-Based Detection Advantages andDisadvantages of peaks. distinguish homozygotes,asinglepeak,from heterozygotes, two distinguished bytheirmeltingpeaks.Thismakesitpossibleto nucleotide willmeltatslightlydifferent temperatures andcanbe be useful for mutation detection as amplicons that differ by a single expected from theprimer pair. Inthissituation,SYBRGreen Imay that containsonlyasinglepeakrepresenting thespecificproduct assay isfullyoptimized,itpossibletoproduce ameltingprofile may containmultiplePCRproduct species.However, ifthePCR note thatthepopulationsare notnecessarilyhomogeneousand temperatures andare observedasdistinctpeaks.Itis importantto Products ofdifferent lengthsandsequenceswillmeltatdifferent derivative of the fluorescence as a function of temperature (-dF/dT). converted todistinctmeltingpeaksbyplottingthefirstnegative as a drop in fluorescence as the dye dissociates. The melt curves are dsDNA. The point at which the dsDNA melts into ssDNA is observed readouts are continuallycollected.Thiscausesdenaturation ofall temperature gradient from about 50 °C to 95 °C while fluorescenceTo produce meltcurves,thefinalPCRproduct isexposedtoa structure, andthechemicalformulationofreaction chemistry. qPCR DetectionMethods analysis usingdsDNAbindingdyes. information abouthighresolution meltanalysisforgenotype

tion ofPCRproducts Are themosteconomicalformatfordetectionandquantifica - 1)  Figure 3.Dye-BasedqPCRusingSYBRGreen IBindingDye presence ofsingle-strandedDNA. has nosignificantfluorescence inthe primers andtemplate.SYBRGreen I dNTPs, buffer andSYBRGreen I, The PCRreaction containsenzyme, 2 Pleaseseewww.corbettresearch.com forfurther sigma.com

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tion willbeevenmore inaccurate. shorter one.Ifamplificationefficiencies are different, quantifica- tion thatthere are more copiesofthelongertemplate thanthe generate more signalthanashorterone,leadingtotheimplica - amplification efficiencies, ofalonger product will mass ofdsDNAinthereaction. Eveninreactions withthesame single amplifiedmolecule.Signalintensityisdependentonthe isthatmultipledyemoleculesmaybindtoa Another concern accurate quantificationwhenusingSYBRGreen Ibindingdye. dramatically reduce non-specific primingandprovide more separate reverse-transcription, PCR,andDNasetreatment can to primerdimersandmisprimedproducts. ForRT-PCR assays, the “notemplatecontrols” (NTC)duetodyemoleculesbinding product. Non-specificbinding results influorescence readingsin in non-specificfluorescence andoverestimation oftheactual (ssDNA). TheyalsobindindiscriminatelytoanydsDNA,resulting Some oftheDNAbindingdyeswillbindtosingle-stranded n n Disadvantages n n n n

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The DNA polymerase then displaces the Reporter molecule from the probe Probe-Based Detection resulting in fluorescence. The fluorescence accumulates as cycling of PCR contin- Probe-based quantitation uses sequence specific DNA-based ues and is measured at the end of each PCR cycle. The intensity of fluorescence generated by the Reporter molecule above background level (Ct value) is mea- fluorescent reporter probes. Sequence specific probes result in

sured and used to quantitate the amount of newly generated double-stranded qPCR Detection Methods quantification of the sequence of interest only and not all dsDNA. DNA strands. The probes contain a fluorescent reporter and a quencher to Replicated DNA: After repeating the denaturation, annealing and extension prevent fluorescence. Common fluorescence reporters include cycles approximately 35-40 times, analysis can begin. The Ct values can be used derivatives of fluorescein, rhodamine and cyanine. Quenching is to quantitate starting amounts of DNA or to establish a standard curve for gene expression studies or other comparative analysis. the process of reducing the quantum yield of a given fluorescence process. Quenching molecules accept energy from the fluorophore Probe-based quantitation can also be used for multiplexing: and dissipate it by either proximal quenching or by Fluorescence detecting multiple targets in a single reaction. Different probes 3 Resonance Energy Transfer (FRET, see Fig. 4). Most reporter systems can be labeled with different reporter fluorophores allowing the utilize FRET or similar interactions between the donor and quencher detection of amplification products from several distinct molecules in order to create differences in fluorescence levels sequences in a single qPCR reaction. See Appendix 2 for an when target sequences are detected. The fluorescent reporter Applications Note on Multiplexing. and the quencher are located in close proximity to each other in order for the quencher to prevent fluorescence. Once the probe Many probe-based detection systems are available and they are locates and hybridizes to the complementary target, the reporter all variations on a single theme with significant differences in and quencher are separated. The means by which they are separated complexity, cost, and the results obtained. Therefore, it is varies depending on the type of probe used. Separation relieves important that the chemistry selected is appropriate for the quenching and a fluorescent signal is generated. The signal is intended application. For an overview of available specific then measured to quantitate the amount of DNA. The main chemistries, see Table 2. advantage to using probes is the specificity and sensitivity they Chapter 3 contains additional information on available probes afford. Their major disadvantage is cost. and primers for specific quantitation to aid in the understanding and selection of the ideal chemistry. Figure 4. Fluorescence Resonance Energy Transfer Table 2. Probe-Based Chemistries and Modifications Probe-Based Chemistries Linear Probes Structured Probes Hydrolysis probes Molecular Beacons Hybridization probes Scorpions™ Probe Modifications Locked Nucleic Acids (LNA) Minor Groove Binders (MGB)

Denaturation: Double-stranded DNA is heated to 94 °C-98 °C. During this period, the double-stranded DNA helix melts open into two single-stranded templates. Annealing: The reaction is cooled to 45 °C-65 °C. Probes labeled with both a Reporter molecule and a Quencher molecule and sequence-specific primers anneal to the single-stranded DNA template. During this cycle, DNA polymerase attaches to the primed template and begins to incorporate complementary nucleotides (dATP, dCTP, dGTP, TTP). This process is very slow because the poly- merase is inefficient at these lower temperatures. Extension: The temperature is raised slightly during the extension cycle to 65 °C-75 °C. The optimal temperature for Taq DNA polymerase is 72 °C. During this phase, DNA polymerase extends the sequence-specific primer with the incorporation of nucleotides that are complementary to the DNA template.

Our Innovation, Your Research — Shaping the Future of Life Science 5 6 Primer and Probe Design Figure 5illustrateshowthese probes work. reduces enzymeprocessivity, shortamplicons are designed. 5’-3’ exonucleaseactivityofthepolymerase.Sincethisalso elongation stepatalowertemperature toensure maximum at 8-10°CabovetheTmofprimersandtoperform elongation from primers. The probe is usually designed to hybridize to ensure thattheprobe hybridizestothetemplateprior cence from thereporter. Reactionconditions mustbecontrolled separating them,andleadstoanirreversible increase influores- releases thefluorophore andquencherintosolution,spatially displaces the5’endofprobe andthendegradesit.Thisprocess double-strand-specific 5’-3’nucleaseactivityoftheTaq enzyme the probe bindstoitduringeachannealingstepofthePCR.The cence isdetected.IfthePCRresults inacomplementarytarget, the probe ispresent, theprobe remains intactandlowfluores- and aquenchertothe3’end.Ifnoampliconcomplementary tary target.Afluorophore isattachedtothe5’end of theprobe to cleavealabeledprobe whenitishybridizedtoacomplemen- exonuclease activityofThermusaquaticus(Taq) DNA polymerase How TheyWork: TheTaqMan methodrelies onthe5’-3’ Developed by:RocheMolecularSystems Also knownasDualLabeledFluorescent Probes (DLFP) orTaqMan Hydrolysis Probes this sectionare FRETbased. common linear probes are described below. All probes included in and theyare extremely simpletodesignanduse.The most secondary structure allowsforoptimumhybridizationefficiency, The majoradvantagesoflinearprobes are thattheabsence of Linear Probes design. LockedNucleicAcids use isalsobeingmadeofmodificationsinprimerandprobe more complexstructure, suchastheScorpions.Finally, increasing method, theMolecularBeacontechnologyandprobes witha Examples includetheSYBRGreen intercalating dyedetection formats availablethatmayoffer advantagesincertainsituations. this methodisthemostwidelyused,there are severalother probe labeledwith5’reporter and3’quenchermolecules.While polymerase used.Theassayrequires apairofPCRprimersand fluorescent signalduetotheinherent nucleaseactivityofthe format. Thisformatfunctionsbyrelease andgenerationofa qPCR assays.Thevastmajorityofassaysusethe5’nuclease Various primerandprobe formatsare availableforperforming Primer andProbeDesign and structured probes. methods availablefallintotwodifferent categories:linearprobes not beasamenabletothemore traditionalapproaches. The (MGB ™ ) provide significantadvantagesincertainassaysthatmay sigma.com ORDER: 800-325-3010 ® (LNAs)andMinorGroove Binders TECHNICAL SERVICE: 800-325-5832 ®

tion applicationsandforthoserequiring multiplexing. Uses: Undoubtedlythechemistryofchoiceformostquantifica- on theactivityofTaq DNApolymerase. Advantage: ThisisthemostpopularqPCRchemistryandrelies complementary amplicon. the probe todissociateatadifferent temperature tothefully is positionedoverthepolymorphic siteandthemismatchcauses Uses: CanbeusedforSNP/mutationdetection, where oneprobe is reversible andallowsforthegenerationofmeltcurves. Advantages: Sincetheprobes are not hydrolyzed, fluorescence (5’-LC Red640)isaprocess knownasfluorescence resonance energytransfer. from thedonorfluorophore (3’-fluorescein) totheacceptorfluorophore a singlestrandofamplificationproduct. Thetransferofresonance energy Hybridization probes produce fluorescence whenbothare annealedto such asLightCycler FAM for example.Theotherhasanacceptordyeonits5’end, one anotherontheamplicon.Onehasadonordyeatits3’end, How TheyWork: Two probes are designedtobindadjacent to with manyreal-time instruments. Technology/Roche capillary-basedinstrument,butcanbeused Developed by:specificallyforusewiththeIdaho Hybridization Probes used togenerate meltcurves. how theseprobes work. light emittedismeasured bythesecondprobe. Figure 6illustrates energy totheacceptordyethrough FRETandtheintensityof during theannealingstep.Thereporter isexcitedandpassesits hybridize tothetargetsequenceinahead-to-tailarrangement to prevent extensionduringtheannealingstep.Bothprobes Figure 5.FunctionofHydrolysis Probes Figure 6.FunctionofHybridization Probes 5’ Primer 5’ ® Red640or705,andisblockedatits3’end Donor Probe bv 4 Asstatedabove, theycanalsobe Acceptor Probe bv P 5’

INNOVATION @ WORK

Structured Probes Uses: Molecular Beacons7 probes have become popular for standard analyses such as quantification of DNA and RNA.8 They Thermodynamic analysis reveals that structurally constrained have also been used for monitoring intracellular mRNA hybridiza- probes have higher hybridization specificity than linear probes.5,6 9 10 11

tion, RNA processing, and transcription in living cells in real- Primer and Probe Design Structurally constrained probes also have a much greater time. They are also ideally suited to SNP/mutation analysis12 as specificity for mismatch discrimination due to the fact that in the they can readily detect single nucleotide differences13,14 and have absence of target they form fewer conformations than unstruc- been reported to be reliable for analysis of G/C-rich sequences.15 tured probes, resulting in an increase in entropy and the free The sensitivity of Molecular Beacons probes permits their use for energy of hybridization. The following outlines the traits of the the accurate detection of mRNA from single cells.16 They have two most common structured probes. been used in fourplex assays to discriminate between as few as Molecular Beacons Probes 10 copies of one retrovirus in the presence of 1 × 105 copies of another retrovirus.17 Developed by: Public Health Research Institute in New York ™ How They Work: They consist of a hairpin-loop structure that Scorpions Probes forms the probe region and a stem formed by annealing of Developed by: DsX, Ltd. complementary termini. One end of the stem has a reporter How They Work: The Scorpions uni-probe consists of a single- fluorophore attached and the other, a quencher. In solution, the stranded bi-labeled fluorescent probe sequence held in a hairpin- probes adopt the hairpin structure and the stem keeps the arms in loop conformation (approx. 20 to 25 nt) by complementary stem close proximity resulting in efficient quenching of the fluorophore. sequences (approx. 4 to 6 nt) on both ends of the probe. The During the annealing step, the probe is excited by light from the probe contains a 5’-end reporter dye and an internal quencher dye directly linked to the 5’-end of a PCR primer via a blocker. PCR instrument (hu1). Molecular Beacons hybridize to their target sequence causing the hairpin-loop structure to open and separate The blocker prevents Taq DNA polymerase from extending the the 5’-end reporter dye from the 3’-end quencher. The quencher PCR primer. The close proximity of the reporter dye to the is no longer close enough to absorb the emission from the quencher dye causes the quenching of the reporter’s natural reporter dye. This results in fluorescence of the dye and the PCR fluorescence. instrument detects the increase of emitted energy (hu2). The At the beginning of the real-time quantitative PCR reaction, Taq resulting fluorescent signal is directly proportional to the amount DNA polymerase extends the PCR primer and synthesizes the of target DNA. If the target DNA sequence does not exactly match complementary strand of the specific target sequence. During the the Molecular Beacon probe sequence, hybridization and next cycle, the hairpin-loop unfolds and the loop region of the fluorescence does not occur. When the temperature is raised to probe hybridizes intra-molecularly to the newly synthesized target allow primer extension, the Molecular Beacons probes dissociate sequence. The reporter is excited by light from the real-time from the targets and the process is repeated with subsequent quantitative PCR instrument (hu1). Now that the reporter dye is PCR cycles. See Figure 7 for an illustration of how these probes no longer in close proximity to the quencher dye, fluorescence work. emission may take place (hu2). The significant increase of the fluorescent signal is detected by the real-time PCR instrument Figure 7. Function of Molecular Beacons Probes and is directly proportional to the amount of target DNA. See Figure 8 for an illustration of how these probes work.

Figure 8. Function of Scorpions Probes

Advantage: Greater specificity for mismatch discrimination due to structural constraints. Disadvantage: The main disadvantage associated with Molecular Beacons is the accurate design of the hybridization probe. Optimal design of the Molecular Beacon stem annealing strength is crucial. Scorpions bi-probe, or duplex Scorpions probes, were developed This process is simplified with the use of specific software packages to increase the separation of the fluorophore and the quencher. such as Beacon Designer from Premier Biosoft or by contacting The Scorpions bi-probe is a duplex of two complementary labeled a member of the design team at www.designmyprobe.com oligonucleotides, where the specific primer, PCR blocker region, for assistance. probe, and fluorophore make up one oligonucleotide, and the quencher is linked to the 3’-end of a second oligonucleotide that

Our Innovation, Your Research — Shaping the Future of Life Science 7 8 Primer and Probe Design human papillomaviruses. detection andhavebeenusedtodetect,typequantitate Uses: Scorpionsprobes are ideallysuitedtoSNP/mutation amplicons generatedandtheamountoffluorescence produced. there isaone-to-onerelationship betweenthenumber of displace andcleavetheprobe. Themostimportantbenefitisthat reduced temperature required forthe5’-nucleaseactivityto optimal temperature forthepolymeraseratherthanat TaqMan assaysisthatthePCRreaction iscarriedoutatthe that containsa2’-O, 4’-Cmethylenebridge.The bridgeislocked A LockedNucleicAcid(LNA)isa noveltypeofnucleicacidanalog Locked NucleicAcid modifications. and MinorGrove Bindersare twoofthemostcommonlyused probes toprovide enhancedperformance.LockedNucleicAcids A numberofmodificationscanbeincorporatedwhendesigning Modifications amplicon ensuringthateachprobe hasatargetinthevicinity. efficient duetocovalentattachmentoftheprobe tothetarget event. Aunimoleculareventiskineticallyfavorableandhighly one moleculeconvertingprimingandprobing intoaunimolecular Advantages: Scorpionsprobes combinetheprimerandprobe in action isessentiallythesameasuni-probe format. is complementarytotheprobe sequence.Themechanismof Primer andProbeDesign and lowsignal/lowbackground ratioofMolecularBeaconsprobes. with thehighsignal/highbackground ratiooftheTaqMan probes allele ofaSNP. ResultswithScorpionsprobes compare favorably the PCRprimercanbedesignedtoselectivelyamplifyonlyone versions oftheprobe with different fluorophores. Alternatively, SNP canbedetectedinasinglereaction bylabelingthetwo sion. Iftheprobe sequenceisallele-specific,allelicvariantsofa either byallelespecifichybridizationorexten- Scorpions bi-probes work. uni-probe format.SeeFigure 9foranillustrationofhow resulting inimproved signalintensityoverthestandard Scorpions arrangement retains theintramolecularprobing mechanism probes andMolecularBeaconsprobes. with asignificantlystronger signalcompared toboth TaqMan This allows introduction of more rapid cycling conditions combined Enzymatic cleavageisnotrequired sothereaction isveryrapid. Figure 9.FunctionofScorpionsBi-Probes sigma.com 21 ORDER: 800-325-3010 SNPdetectioncanbecarriedout 20 Anotheradvantageover TECHNICAL SERVICE: 800-325-5832 18 This 19

exceptional biologicalstability. This structure confersenhancedhybridizationperformanceand nose ringandlockingthestructure intoarigidbicyclicformation. in 3’-endoconformationrestricting theflexibilityofribofura- n n n n n Advantages inaccessible SNPs,suchastherelatively stableG:Tmismatch. GC-rich regions. LNAsalsofacilitatethequeryingofdifficult or address traditionallyproblematic targetsequences,suchasAT- or eliminated. Forinstance,shorter probes canbedesignedto be overcome withstandard DNAchemistriescanbereduced or design guidelines.Assuch,certainlimitationsthatcannot be shorter, allowingflexibilityindesignwhilestillsatisfyingassay contribution, LNAcontainingqPCRprobes canbesynthesizedto Due totheenhancedhybridizationcharacteristicsandTm Added FlexibilityinProbe Design conventional DNAprobes. ing LNAbasescanbeusedatthesametemperatures aslonger conventional DNAfluorescent probe. Shorterprobes incorporat- LNA fluorescent probe anditstargetnucleicacidthanwitha greater destabilizingeffect ontheduplexformationbetweena ing applications.Thepresence ofasinglebasemismatchhas extremely reliable andeffective meansforSNP-callingingenotyp- LNAs canalsobeusedforallelicdiscrimination.Theyprovide an Enhanced AllelicDiscrimination on thetargetnucleicacid. DNA fluorescent probe forthesametargetsequence, depending monomer substitutioninmediumsaltconditionscompared toa melting temperature, Tm,mayincrease byupto8°C perLNA the hybridizationofprobe toitstarget.Asaresult, theduplex The LNAmonomerchemicalstructure enhancesthestabilityof Increased StabilityandImproved Specificity base(s) intheprobe design. and thusthehybridizationspecificityviaplacementofLNA ally, thisbenefitalsomakesitpossibletooptimize theTmlevel allows formore successfulsingle-tubemultiplexing. a significantbroadening inthescopeofassayconditionsand reduced andthesignal-to-noiseratioisincreased. specificity, background fluorescence from spuriousbindingis

Compatible withmanysystems Added flexibilityinprobe design Enhanced allelicdiscrimination Improved specificity of probe hybridization to target sequence Increased thermalduplexstability LNA Monomer HO O O 24 Base 26 Thisincrease inhybridizationcreates Byincreasing thestabilityand 22 HO O O Base 25 Addition- 23 INNOVATION @ WORK

LNAs can also be used when AT-rich qPCR probes need to be over Template, Primer Design, 30 bases long to satisfy amplicon design guidelines. With LNA fluorescent probes, the selective placement of LNA base substitu- Probe Design and Dye Choice

tions facilitates optimal design of highly specific, shorter probes Primer and Probe Design It is very important to ensure that the primers and probes are that perform very well, even at lengths of 13-20 bases. optimally designed in order to perform successful quantitative Compatible with Many Platforms PCR. There are many points in the design process that must be LNA probes are also compatible with real-time PCR platforms and considered: the template, primer design, probe design, dye end-point analytical detection instruments depending on the choice in probe design, quenchers in probe design, and the excitation/emission wavelengths of the dyes and of the equip- software the system uses. ment. This provides the ability to work with the instrumentation Template Considerations and reagents of choice under universal cycling conditions. 1. Amplicons should ideally be between 50-150 bases in length. Minor Groove Binders Shorter amplicons tend to be more tolerant of less than ideal reaction conditions and allow probes to successfully compete Minor Groove Binders (MGBs) are a group of naturally occurring with the complementary strand of the amplicon. This allows antibiotics. They are long, flat molecules that can adopt a crescent for faster and more efficient reactions and increased consis- shape allowing them to bind to duplex DNA in the minor groove. tency of results. MGBs are stabilized in the minor groove by either hydrogen bonds 2. For RNA targets, select primers spanning exon-exon or hydrophobic interactions. This results in the stabilization of the junctions. probe-DNA hybridization. By choosing primers that span exon-exon junctions, amplifica- MGBs produce a “Tm leveling” effect as A/T content increases. tion of contaminating genomic DNA in cDNA targets can Natural 8-mer probes exhibit a linear decrease in Tm as A/T be avoided. content increases while MGB probes exhibit level Tm as A/T 3. Consideration of template secondary structure is content increases. The increase in melting temperature allows for important. the use of shorter probes with improved mismatch discrimination. Close attention should be paid to template secondary structure. Advantages This is one area where qPCR primer and probe design differs n Useful for functional assays for difficult targets slightly from traditional PCR design. In traditional PCR design, n Higher accuracy and confidence in results template secondary structure is not necessarily a critical factor. Templates containing homopolymeric stretches of greater than n Compatible with any real-time PCR detection system four consecutive bases should be avoided. Significant second- Functional Assays for Difficult Targets ary structure may hinder primers from annealing and prevent complete product extension by the polymerase. This is a There are many sequence features and applications that pose particular concern in cases of stretches of Gs. A useful tool for problems when trying to design probes and primers for specific the assessment of template secondary structure in DNA targets targets. For example, sequential “Gs” result in secondary is the Mfold server, developed by Dr. Michael Zuker and structure that can block efficient hybridization, weaker bonds maintained at the Rensselaer and Wadsworth Bioinformatics within “A/T” rich regions result in lower melting temperatures Center. (http://www.bioinfo.rpi.edu/applications/mfold/) (Tm) that inhibit the use of efficient PCR conditions, and limited target regions can restrict the length of optimal probe and primer Primer Design Considerations sequences impacting their functionality. Since MGBs allow for 1. Avoid secondary structure problems. design of shorter probes and for the design of specific Tms, they When feasible, one should attempt to pick regions of the aid in the success of assays posing these difficulties. template that are not apt to cause secondary structure Higher Accuracy and Confidence in Results problems. For more information, please refer to number 3 under “Template Design Considerations.” Since shorter probes can be used, mismatch sensitivity can be increased with the use of MGBs. Also, since MGB probes have 2. Avoid runs of Gs and Cs longer than three bases. more stable hybridization characteristics and remain intact after In the primer sequences, runs of Gs and Cs longer than three PCR, they are not cleaved by the 5’-nuclease activity of Taq DNA bases should be avoided. This is very important at the 3’-end polymerase. As a result, more symmetric melting curves using the of a primer where such runs may result in a phenomenon same probe and primer set in the same tube as the real-timer PCR referred to as polymerase slippage, which can be a problem in experiments can be produced to confirm PCR and detection specificity. standard PCR. Polymerase slippage occurs during replication Compatible with Any Real-Time PCR Instrument when Taq polymerase slips from the DNA template strand at the repeat region and then reattaches at a distant site. This can MGB Eclipse probe systems will work on any manufacturer’s real- cause a new DNA strand to contain an expanded section of DNA. time or end-point analytical instrumentation. The probes have been tested on most leading systems.

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Primer and Probe Design 2.  1.  Probe DesignConsiderations 7.  6.  5.  4.  3.  Primer andProbeDesign during mismatch detection.Insuchcases,theuse ofspecial probes canbeused.Theuseofshorterprobes isimportant the primers.Whenprobes annealbefore theprimers,shorter reaction. Duringthereaction, theprobe should annealbefore This allowsforefficientprobe-to-target annealingduring the mately 10ºChigherthanthe Tm oftheprimers. In mostsituations,theprobe Tmshouldbeapproxi- perhaps upto35bases. the self-annealinghairpindesign,probes maybealittlelonger, Molecular Beaconsprobes, where quenchingisafunctionof compromise theefficiency ofsignalquenching.Inthecase length tendstorangeup30bases.Longerprobes might probes withareporter andaquencher, theoverallprobe In thecaseof5’-nucleaseassayusingdual-labeledprobes, Probes maybeanywhere from 9-40basesinlength. same annealingtemperature. Multiplexed primerpairsmustallworkefficientlyatthe resources are availabletoaidinthisprocess. should beavoided.Anumberofcommercial primeranalysis In particular, 3’self-complementarityprimer-dimer formation self-complementarity intheirsequences. Forward andreverse PCRprimersshouldbeanalyzed for available athttp://www.ncbi.nlm.nih.gov/sutils/e-pcr/. PCR primersusedtogeneratetheknownSTSs.Thistoolis identify primersthathavetheproper order thatcould represent human genome.e-PCRusesUniSTS,adatabaseofSTSs,to known and,asaresult, theycanserveaslandmarksinthe in thehumangenome.Theirexactlocationandsequenceare the PCRprimers.STSsare shortDNAsegmentsoccurringonce sequences bysearching forsub-sequencesthatcloselymatch e-PCR is used to identify sequence tagged sites (STS) within DNA sequences. Anothermethodistouseelectronic PCR,ore-PCR. compares primersequencesagainstalibraryofgenomicDNA NCBI athttp://www.ncbi.nlm.nih.gov/. ABLASTsearch performed usingtheresources ofthepubliclyavailable siteat BLAST, orBasicLocalAlignmentSearch Tool, searches can be carrying outaBLASTsearch. Check theoverallspecificityofprimers(andprobes) by completely known. plasmid, itisnotalwaysthecasethatsequence predictable targetsequences,suchasplasmids.Evenwitha DNA, andshouldbeguarded againstevenwhenusingmore more markedintargetsofhighercomplexity, suchasgenomic specific, primerbinding.Suchrandombindingmaybe Shorter primersincrease thechanceofrandom,ornon- Design primerslongerthan17bases. resource inthisphaseofthedesignprocess. secondary structures. Again,theMfoldserverisaveryuseful Tm, higherthantheTmofanypredicted template should alsoaimtodesignprimerswithameltingtemperature, have beenunfoldedbefore theprimer-annealing step,one To besure thatthemajorityofpossiblesecondarystructures any ofthepredicted templatesecondarystructures. Aim todesignprimerswithaTmhigherthantheof sigma.com ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

5.  4.  3.  Table 3.DyeChoiceinProbe Design wavelength forseveralpopulardyesusedinprobe design. Table 3provides theexcitationwavelengthandemission 3.  2.  1.  probes. It isalsoimportanttoconsiderthedyechoicewhendesigning Dye ChoiceinProbe Design releases theenergyfrom thequencherasfluorescence atalonger acceptor canbeanotherfluorophore, inwhichcasethetransfer linear probe shouldnotexceedapproximately 30bases.The maximum distancebetweenareporter anditsquencheron a by about100Å.Sinceahelixoccupies approximately 3.4Å, the FRET occurswhendonorandacceptor moleculesare separated Quenchers inProbe Design Cy5 ROX/Texas Red TAMARA Cy3 HEX/JOE/VIC TET FAM/SYBR Green Alexa 350 Filter This mayleadtospontaneousfluorophore quenching. to thereporter dyeshouldbeavoided. A Gplacedattheextreme 5’endoftheprobe adjacent to annealinthemiddleofamplicon. For MolecularBeaconsprobes, theprobe shouldbedesigned forward primer. should beasclosepossibletothe3’-endof 5’-terminal oftheprobe (carryingthereporter dye) For optimalefficiencyofdual-labeledprobes, the runs ofsinglebases,mostdefinitelynotGs,inprobes. As inthecaseofprimers,there shouldnotbehomopolymeric preferably withagreater numberofCsthanGs. Probe GCcontentmaybeanywhere from 30-80% atic targetsequences. providing easierandmore flexibleprobe designsforproblem- more accurategenequantitationandallelicdiscrimination the thermalstabilityandhybridizationstability, allowingfor complementary DNAandRNA.LNAprobes servetoincrease RNA nucleotidesthatexhibitthermalstabilitiestowards probes maybebeneficial.LNAfluorescent probes are modified modifying bases,orLockedNucleicAcid should beminimized. When designingmultiplexedreactions, spectraloverlap sure thatthefluorophore andquencherare compatible. When designingfluorescent probes, itisimportant tobe the dyes. Make sure thattheinstrumentbeingusedcandetect Excitation Wv 635 585 556 545 535 517 492 350 ® (LNA Emission Wv ® ) fluorescent 665 610 580 568 555 538 516 440 INNOVATION @ WORK

wavelength. For instance, the combination of FAM (a fluorescein Primer and Probe Design FAQs derivative) and TAMRA (a rhodamine derivative) will absorb at What type of probe should I choose? 492 nm (excitation peak for FAM) and emit at 580 nm (emission It depends on the experiment being performed and the machine peak for TAMRA). The inherent fluorescence and broad emission being used. Primer and Probe Design spectrum of TAMRA result in a poor signal-to-noise ratio when this fluorophore is used as a quencher. This can make multiplexing Table 5 is intended to aid in the selection of probes based on the difficult. To address this issue, dark quenchers were introduced. desired application. Dark quenchers absorb the energy emitted by the reporter Table 5. Probe Applications fluorophore and emit heat rather than fluorescence. Early dark TaqMan/ Molecular quenchers, such as 4-(49-dimethylaminophenylazo) benzoic acid Application Hydrolysis Hybridization Beacons Scorpions (DABCYL), had limited spectral overlap between the fluorescent dye and quencher molecule. Black Hole Quenchers (BHQ™-1 and Quantification X X BHQ™-2) from Biosearch Technologies have lower background applications fluorescence and a broad effective range of absorption. As a Multiplexing X X result, Black Hole Quenchers provide greater sensitivity and SNP/mutation X X X enable the simultaneous use of a wide range of reporter detection fluorophores thus expanding the options available for multiplex Generation of X assays. It is important to ensure that the Black Hole Quencher is melt curves matched with the probe based on the excitation and emission spectra of the probe. Monitoring X intracellular Table 4. Quencher Ranges mRNA Quencher Quenching Range hybridization, RNA processing, BHQ-1 480-580 nm and transcription BHQ-2 550-650 nm in living cells BHQ-3 620-730 nm Analysis of G/C- X TAMARA 550-576 nm rich sequences DABCYL 453 nm Detection of X mRNA from Primer and Probe Design Software and Web Sites single cells Several software programs can be used to design primers and Why do I need to have a good design? probes, but software is only a tool to aid in the design process Well-designed primers and probes will provide ideal RT-qPCR and cannot guarantee a perfect design. data: high PCR efficiency, specific PCR products and the most sensitive results. Designing Primers What should I do if no primers and/or probes are found for n Primer 3.0 my sequence? Designing TaqMan Probes or Molecular Beacons Probes There are several parameters that can be adjusted to force the n Beacon Designer from Premier Biosoft program to pick primers and/or probes without significantly sacrificing primer and/or probe quality. Designing Scorpions Probes I have a very short target sequence. Is it possible to design n DNA Software at dnasoftware.com an optimal probe? Designing FRET Probes (LightCycler Probes) It depends on the sequence. To design the probe, it may be n LightCycler Probe Design Software 2.0 necessary to submit a longer target sequence (up to 160 bases). Additionally, LNA bases can be included in the probe sequence to Designing LNA Probes or Oligos reduce the probe length while retaining the optimal characteristic n A combination of different types of software must be used of the probe.

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Instrumentation other fluorescent dyeinthe reaction. signal from onefluorescent dyeisdetectedassignalfrom the termed “cross talk.”Cross talkoccursinmultiplexreactions when neighboring reactions onthe plate.Onesignificantexampleis from thedetectionchemistry andnotfromsources alternative or it isessentialthatthesignaldetectedgeneratedspecifically Specificity of detection is important at the single dye level because Specificity ofDetection systems usingexactlythesametargetanddetectionchemistry. As aresult, itisimportant to carryoutcomparisonsbetween not onlyinstrumentdependent,butisalsochemistrydependent. detected abovethebackground noiseofthesystem.Sensitivityis Sensitivity refers totheminimum quantityoftargetthatcanbe Sensitivity detection linearity. system are sensitivity, specificityofdetection,dynamicrangeand debated, thecriticalfactorstoconsiderwhenselectingaqPCR While themeritsofonehardware designoveranother canbe tubes (PMTs) asphotodetectors. coupled device(CCD)cameraswhileothersusephotomultiplier Many companieshavecontinuedtousesilicon-based,charge- light filters. bulbs orlightemittingdiodes(LEDs),inconjunctionwithspecific resolved byusingbroad wavelengthexcitationsources, tungsten dyes for multiplexing. The cost and application disadvantages were single dyebutdoesnotallowtheflexibilitytoincludeadditional single wavelengthallowsforthespecificityofexcitationa tage, ofusinglasersisthattheyemitatasinglewavelength.A source beingthelaser. Themajoradvantage,andalsothedisadvan- excitationsourcesAlternative were alsodeveloped,withthefirst to reduce thiseffect. manufacturers investedinuniquethermalregulation mechanisms this situationiscontrarytotheneedsofquantitativePCR,many caused differences inPCRefficiencyandproduct quantity. Since uniformity thatresulted inthe“edgeeffect.” Lackof uniformity Conventional PCRthermalblockswere notoriousforalackof used forquantitativestudies. components ofthesystemrequired specifictraitsinorder tobe During instrumentdevelopment,itbecameclearthatcertain Components When Higuchiet.al Considerations Instrumentation Detection Linearity;Software. Components; Sensitivity;SpecificityofDetection;DynamicRange; it isimportanttoconsiderthefollowingtraitsofsystem: mentally thesame.WhenchoosinganinstrumentforqPCR, tation, buttherequirements forasystemhaveremained funda- instrument, there havebeenmanyadvancesinqPCRinstrumen- were theinstrumentsused.SincedesignoffirstqPCR PCR block,aUVlightsource andacameraforsignaldetection sigma.com 1 designedaqPCRsystem,conventional ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

6. graphics packageswhileotherssimplylistCtvalues. market offerings. Somemanufacturers dependoncommercial in whichdataispresented variesconsiderablyamongthecurrent additional considerationisthepresentation ofdata.Themanner fluorescent thermocyclerasathermostatedfluorometer. An fluorescence datawouldbeuseful,asinthecaseofusinga analysis choices.Asecondconsiderationiswhetherhavingraw data isaccessibleandthescientistchoosesamongextensive are fullyflexible.Inthecaseoffullflexibility, everypointofraw little, ifany, inputfrom theenduser. Incontrast,otherpackages Some packagesare targetedtowards simplicityofanalysiswith dependant uponthecapabilitiesofintegratedsoftware. For manyscientists,thechoiceofsystemwillbelargely Software is abletolinearlymeasure. Detection linearityistheconcentrationrangethataninstrument Detection Linearity analyses capabilities. determined bythephotodetectoraswellsoftware is restricted bychemistryandtherangeofsignaldetectionis signal-to-noise discriminationofthesystem.Themaximumsignal The minimumdetectablesignalisdeterminedbychemistryand minimum andmaximumtargetconcentrationsthatcanbedetected. The dynamicrangeofdetectionisthedifference betweenthe Dynamic Range 5. 4. 3. 2. 1. systems, itisimportanttoperformthefollowingtests: the sametargetanddetectionchemistry. When comparing As statedabove,itisimportanttocompare systemsusingexactly Comparing qPCRInstrumentation a.  Wavelength Discrimination a.  Dynamic Range–Reaction a.  Dynamic Range–Optical a.  Uniformity–Thermal andOptical b.  a.  Uniformity–Optical a. Uniformity–Thermal intensity from afluorophore inamixture offluorophores. This istheabilitytoaccuratelymeasure fluorescence 10 Perform qPCRonaseriallydiluted template(1copyto also beusedasfluorescent microplate readers. cence. Thisspecificationis relevant toinstrumentsthatcan approximated bytheminimum above-background fluores- significant, qPCRisbaseduponCtmeasurement, whichis is importanttonotethatwhileopticaldynamicrange Plot fluorescence measurements ofaserialdyedilution.It Ct values. Run identicalqPCRexperimentsineverywellandcompare probes allocatedtoeverywell oftheplate. Compare thestartingfluorescence levelsforidentical qPCR aliquoted toeverywell. Measure thefluorescence ofidenticalconcentrations ofdye Run acalibratedend-pointPCRanddetectongel. 12 copies).

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On the following pages, qPCR instruments are categorized into more will be considered high throughput. All qPCR instruments two groups: high throughput and low throughput. Any device require a computer for operation and data management/analysis. that processes fewer than 96 samples in a run will be called Nearly all instruments are supplied with a computer and the

low throughput and anything that processes 96 samples or appropriate software. Instrumentation

High-Throughput qPCR Instruments Applied Biosystems Applied Biosystems Applied Biosystems Roche Applied Science Manufacturer ABI 7300 Real-Time ABI 7500 Real-Time ABI 7900HT Fast LightCycler® 480 System PCR System PCR System Real-Time PCR System Real-Time PCR System Advantages Affordable, 5 color multiplexing. Rapid 96- and 384-well plate Interchangeable 96- and 4 color multiplexing cycling upgrade available compatible. The system 384-well thermal blocks. in the ABI 7500 Fast that can handle TaqMan Low The Therma-Base design allows run times of <40 Density Arrays with fully provides added tempera- min. TaqMan assays are automated robotic ture uniformity available loading. Optional Fast real-time PCR capability. TaqMan assays are available Excitation Tungsten-halogen lamp, Tungsten-halogen lamp, Extended-life 488 nm Filtered (450, 483, 523, single-excitation filter 5 excitation filters argon-ion laser is 558 and 615 nm) high- distributed to all wells by intensity Xenon lamp a dual-axis synchronous (430–630 nm) scanning head Detection 4 emission filters and a 5 emission filters and a CCD camera and a Filters (500, 533, 568, CCD camera CCD camera spectrograph 610, 640, 670 nm) and a CCD camera Heat block Peltier Peltier Peltier Peltier plus Therma-Base Rxn volume(s): 20-100 µL 25-100 µL 25-100 μL/10-30 μL (fast) 5-100 μL (96 well) or for 96-well and 5-20 μL 5-20 μL (384 well) for 384-wells Multiplexing Up to 4 fluorophores Up to 5 fluorophores Multiplex number is The number is dependent limited by spectral overlap on the excitation/emission combination Sensitivity 10 starting copies of the 10 starting copies of the 10 starting copies of the 10 copies (plasmid) RNase P gene from human RNase P gene from human RNase P gene from human genomic DNA genomic DNA genomic DNA Dynamic Range– 9 9 9 10 Reaction – Orders of Magnitude Software System Software and System Software and System Software and LightCycler Software Primer Express software Primer Express software Primer Express software included included included. Enterprise Edition Software with SNP Manager and RQ Manager are available

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Instrumentation High-Throughput qPCRInstruments Instrumentation Software of Magnitude Reaction –Orders Dynamic Range– Sensitivity Multiplexing Rxn volume(s): Heat block Detection Excitation Advantages System Manufacturer sigma.com qPCR System MxPro 10 To asingle-copyequivalent Up to4fluorophores 10-60 µL Peltier photomultiplier tube position filterwheelanda Scanning fiberopticswithfour- Quartz tungsten-halogenlamp applications from asinglePC same timeforhigh-throughput multiple systemstobeusedatthe also belinkedtoaPCallowing safeguards data.Thesystemcan acids). Anembeddedcomputer fluorescently stained/labelednucleic microplate reader (e.g.,toquantify as athermostatedfluorescent Red probes. Systemcanbeused allowing theuseofROXorTexas A reference dyeisunnecessary Stratagene Mx3000P ORDER: 800-325-3010 ™ included ® Multiplex TECHNICAL SERVICE: 800-325-5832 MxPro Stratagene Mx3005P Beacon Designer4software 10 To asingle-copyequivalent Up to5fluorophores 10-60 µL Peltier photomultiplier tube position filterwheelanda Scanning fiberopticswithfour- Quartz tungsten-halogenlamp applications from asinglePC same timeforhigh-throughput multiple systemstobeusedatthe also belinkedtoaPCallowing safeguards data.Thesystemcan acids). Anembeddedcomputer fluorescently stained/labelednucleic microplate reader (e.g.,toquantify as athermostatedfluorescent Red probes. Systemcanbeused allowing theuseofROXorTexas A reference dyeisunnecessary qPCR System ™ included.Alsoincludes ® Multiplex Quansoft software included 9 to 1nMfluorescein Single copyoftemplateanddown Up to4colors 15-50 µL (between 20-70°C) gradient rangeofupto30°C Gradient blockoptionwitha Photomultiplier tube(500-710nm) (470-650 nm) Filtered solid-statewhitelight block loadingmechanism system hasarobot-friendly CD-type controlled from asinglePC.The Up to4instrumentscanbe Techne Quantica ® INNOVATION @ WORK

High-Throughput qPCR Instruments Bio-Rad Laboratories Bio-Rad Laboratories Bio-Rad Laboratories MyiQ™ Single-Color DNA Engine Opticon 2® Bio-Rad Laboratories Manufacturer iQ5 Real-Time PCR Real-Time PCR Real-Time PCR Chromo4™ Real-Time Instrumentation System Detection System Detection System Detection System Detector Advantages Optical upgrade to the Optical upgrade to the Can perform plate reads Has a 96-well Alpha unit. iCycler® thermal cycler. iCycler thermal cycler. This allows it to be used Embedded tool for end- Limited to one color on any DNA Engine® point fluorescence analysis chassis Excitation Tungsten-halogen lamp Tungsten-halogen lamp 96 LEDs (470-505 nm) 4 LEDs in photonics (475-645 nm) (475-645 nm) shuttle (450-650 nm) Detection 5 color customizable 12-bit CCD camera Two photomultiplier 4 photodiodes filter wheel and CCD (515-545 nm) tubes; one compatible (515-730 nm) camera (515-700 nm) with SYBR Green I or FAM; the other detects a variety of fluorophores Heat block Peltier and Joule Peltier and Joule Peltier Peltier (gradient: 25 °C max) (gradient: 25 ºC max) (gradient: 24 ºC max) (gradient: 24 ºC max) Rxn volume(s): 15-100 µL 15-100 µL 10-100 µL 10-100 µL Multiplexing Up to 5 colors No Up to 2 colors Up to 4 colors Sensitivity Linear to 10 copies of One copy of IL-1b in As little as one starting As little as one starting b-actin DNA human genomic DNA template copy template copy Dynamic Range– 9 8 10 10 Reaction – Orders of Magnitude Software iQ5 Optical System iCycler iQ Software DNA Engine Opticon 2 Chromo4 Software Software Version 2.0 Version 3.1 Software Version 3.1 Version 3.1

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Instrumentation Low-Throughput qPCRSystems Instrumentation Software samples number of Maximum of Magnitude Reaction –Orders Dynamic Range– Sensitivity Multiplexing Rxn volume(s): Heat block Detection Excitation System Manufacturer Advantages sigma.com SmartCycler units toasinglePC of 96bylinkingadditional of 16–toatotalnumber be increased in increments Number ofsamplescan 8 10 copiesplasmid Up to4colors 25 and100µLvolumes forced-air cooling Solid-state heater; light used tocollectfiltered Silicon photodetectoris LED SmartCycler Cepheid reaction tubes require proprietary 20 minutes.Fastruns complete inaslittle protocols. Fastprograms pendent experimental programmed forinde­ reaction tobeseparately total, andallowseach of 16,upto96samples Runs samplesinblocks ORDER: 800-325-3010 ® TD System TECHNICAL SERVICE: 800-325-5832 Corbett systemsoftware rotors in specificallydesigned Up to36or72samples Unpublished Unpublished four channel(3000) Two-channel (3000A)or 20-25 µl Thermostated air photomultiplier tube A seriesoffiltersanda visible spectrum 4 LEDsexciteentire reagents using conventional cycling conditionspossible Conventional orrapid between reactions. a uniformtemperature environment toproduce a changingthermal Samples are rotated in any real-time chemistry. designed toworkwith Centrifugal systems Rotor-Gene Corbett Research ™ 3000/3000A and statisticallicense with analysis,graphing Corbett systemsoftware heat-sealed plates 72 individualtubesor Unpublished whole humangenome amplification from a Single-copy genetarget 2, 5,or6coloroptions is typical 5 µLto100µL,but20 Thermostated air photomultiplier tube per channelanda Separate emissionfilter intensity LEDperchannel Separate colorhigh- analysis software reagents. Dedicated using conventional cycling conditionspossible Conventional orrapid between reactions. a uniformtemperature environment toproduce a changingthermal Samples are rotated in any real-time chemistry. designed toworkwith A centrifugalsystem Rotor-Gene Corbett Research ™ 6000 LightCycler 32 10 1-10 copiesofplasmid of humangenomicDNA. Single-copy genein3pg between 530-705nm measures fluorescence Provides multiplexingand 20 µLand100 Thermostated air 710 nm) 610, 640,670and Photodiodes (530,560, Blue LED(470nm) glass capillaries place inpassivated Rapid reactions take variety ofchemistries. based andworkwitha instruments are rotor- The LightCyclerseriesof LightCycler Roche AppliedScience ® Software 4.05 ® 2.0 INNOVATION @ WORK qPCR Applications Guide

The popularity of quantitative PCR lies in the many applications Relative Quantitation for which it can be employed. Common applications include the Relative quantitation requires calculation of the ratio between the detection of single nucleotide polymorphisms (SNPs), measure- amount of target template and a reference template in a sample. ment of DNA copy number, determination of genomic allelic The advantage of this technique is that using an internal standard qPCR Applications Guide variation, diagnosis and quantitation of bacterial and viral can minimize potential variations in sample preparation and infections and the validation of gene expression data from handling. The accuracy of relative quantitation depends on the microarray experiments. In order to perform successful experi- appropriate choice of a reference template for standards. ments using qPCR, the quality of the template and the level of Variability of the standard will influence the results and so it is the control must be considered. important that the appropriate standard is used.27 Some Quality of the Template researchers choose not to run a standard curve and to report target quantities as a fraction of the reference, a technique Quantitative PCR can only be used reliably when the template of termed comparative quantitation. Alternatively, one may assume interest can be amplified and when the amplification method is that the amplification efficiencies of the target and reference are without error. As a result, quality of the template is critical to negligible and quantify the target based solely on the standard success. PCR will only amplify DNA or cDNA with an intact curve determined for the reference sequence. Finally, in the most phosphodiester backbone between the priming sites. In addition, accurate of the relative quantitation techniques, the amplification DNA containing lesions that affect the efficiency of amplification, efficiencies of both the reference and target are measured and a such as abasic sites and thymine dimers, will be either under- correction factor is determined. This process, termed normaliza- represented or may not be represented at all in qPCR. In order tion,27 requires examining a sample containing known concentra- to achieve the best possible quantitation, it is of paramount tions of both target and reference and the generation of two importance that the highest quality nucleic acid template goes standard curves. Since this method measures the amount of into the qPCR. One factor that may affect the quality of the target relative to a presumably invariant control, relative qRT-PCR template is the inclusion of additives that may degrade dNTPs, is most often used to measure genetic polymorphism differences primers, and PCR substrates. Some additives can also directly between tissues or between healthy and diseased samples. It is inhibit the DNA polymerase thus affecting the results of the important to note that if using SYBR binding dye-based systems, qPCR. In addition to using quality template, it is also important the target and internal reference quantitation must be performed to include a set of rigorous controls with every experiment to in separate reactions. ensure success. Levels of Quantitative PCR Controls Qualitative Analysis In contrast to quantitative analysis where each cycle (log-linear The complexity and rigor of the controls required depends on the phase) of the PCR is analyzed, qualitative PCR is an end-point degree of quantitation demanded for the reaction. There are analysis after the PCR has reached plateau phase. Qualitative three levels of qPCR quantitation and therefore three levels of analysis is not as accurate as quantitative; however, it can provide complexity for appropriately controlled reactions. The levels of rapid and adequate information for some experiments. Qualita- quantitation are: absolute quantitation, relative quantitation and tive analysis is gel-based and provides a visual assessment of non-quantitative or qualitative. primer design, specificity and quality of the PCR amplification. Absolute Quantitation Summary Absolute quantitation is the most rigorous of all quantitation qPCR is a powerful and flexible technique, but the results achieved levels. Absolute quantitation requires the addition of external using this method are only valid if the appropriate controls have standards in every reaction to determine the absolute amount of been included in the experiment. The level of required controls the target nucleic acid of interest. To eliminate potential differ- depends on the level of quantitiation desired: absolute, relative ences in quantitation due to annealing, the primer binding sites or qualitative. Once that level of control has been identified, of the external standards must be the same as those in the target employing it when performing qPCR will contribute to the sequence. The ideal external standard contains sequences that success of the experiment. are the same as the target sequence or which vary slightly from the target sequence. Equivalent amplification efficiencies between the target and the external standard are necessary for absolute quantitation. Once a suitable construct or amplicon is identified, a standard curve of external standard dilutions is generated and used to determine the concentrations of unknown target samples.

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Optimizing qPCR in thelast3bases,andanAorT atthe3’-end(Fig.10A). primers thathave2GorCresidues inthelast5bases,1G orC avoid strong 3’-terminal dimers while maintaining specificity,dimer shouldbeunstableaswell(DG≥–6.0kcal,Fig.10C).To choose 3’-end (5’-overlap, Fig. 10B) should be avoided. TheAny primerwithbothaterminalD strongest overall partner mustbenon-existentorveryweak(D dimers formedbyeitherprimerhybridizingwithitselforits design software toanalyze duplexformation.Any3’-terminal To checkthepotentialforprimer-dimer formation,useprimer most especiallywithSYBRGreen dye-baseddetection. primers thatare likelytoform primer-dimers shouldbeavoided, will bedetectedalongwiththedesired product. Asaresult, primer-dimers alsoaffect assayspecificitybecausetheprimers SYBRGreen dye-baseddetection, dye-based detection.With primer-dimers are anissueinbothprobe-based andSYBRGreen thereby reducing assayefficiencyandsensitivity. Therefore, components are divertedfrom synthesisofthedesired product, primer-dimer products are produced andamplified,the reaction true forprimerswithcomplementarityattheir3’-ends.When independent products knownasprimer-dimers. Thisisespecially to primerextensionduringPCRandtheformationoftarget- The propensity ofprimerstohybridizeoneanothermaylead Check PrimerDesignforPrimer-Dimer Potential n n n n n n reaction helpingtoensure successfulquantitation: the followingpreliminary stepswillaidintheoptimizationof Regardless ofwhetherthetargetisDNA(qPCR)orRNA(qRT-PCR), qPCR andqRT-PCR Guidelines forOptimizingBoth results withmaximalspecificityandsensitivity. experiment, theresearcher willbeensured ofvalid,reproducible selectivity. Byproperly optimizingtheconditionsforqPCR are multiplexreactions, astheseoftenrequire bothsensitivityand or viralquantification require highspecificity. Mostchallenging of rare mRNAs require high sensitivity. Assays such as SNPsensitivity. detection Forinstance,pathogendetectionorexpression profiling many delicateqPCRassayswhichrequire maximalselectivityand assays willnotrequire completeoptimization,there are agreat PCR andappropriate settingofthethreshold value.Whilesome the accuracyofqPCRdependsonproper optimizationofthe No matterwhetheryourquantitationistobeabsoluteorrelative, Optimizing qPCR

Set thethreshold value Prepare ameltcurve Validate performancewithastandard curve Optimize probe concentration Optimize primerconcentrations Check primerdesignforprimer-dimer potential sigma.com ORDER: 800-325-3010 G ≥ –2.0 kcal and an extendable G ≥ –2.0 kcal, Fig. 10A). –2.0kcal,Fig.10A). TECHNICAL SERVICE: 800-325-5832

to optimizeprimerandprobe concentrations. over therangeoftargetexpectedinsamples,itisnotnecessary section. Ifdetectionislinearandefficiencygreater than85% Conduct astandard curve analysis, asdescribedinthenext dye-based detectiontominimizenon-specificamplification. levels, 200-400nM,are usually betterwhenusingSYBRGreen maximum sensitivityisnotrequired. Somewhatlowerprimer probe at250nM,especially ifthePCRtargetisabundantand with finalconcentrationsofbothprimersat500nMandthe Satisfactory results forprobe-based qPCRare oftenobtained Optimize PrimerConcentrations at the3’-endandthoseinChybridizetoostrongly overall. only formweakduplexes.Ontheotherhand,thoseinBhybridizetoostrongly are notexpectedtoformsignificant amountsofprimer-dimer becausethey 5.0 PrimerDesignSoftware. Asdescribedinthetext,primersshownA Primer sequenceswere analyzed fortheirabilitytoformduplexesusingOligo B. Unacceptable primers: Unacceptable B. primers: Acceptable A. C. Unacceptable primer: Unacceptable C. Figure 10.AnalysisofPrimersforPrimer-Dimer Potential Ov 3’-dim Ov erall dimers erall dimer & ex 3’-dim ers > -2 kcal/mol -2 > ers tendable 3’-en tendable ers ers < > > < < -6 kcal/mol -6 -2 kcal/mol -2 -6 kcal/mol d

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For maximum sensitivity, optimum primer concentrations must be 3. Aliquot 26 µL master mix into all wells in the PCR plate that determined empirically. Primer concentrations are most efficiently contain primers (A1-E5). optimized by testing various combinations in qPCR, as shown in 4. Mix thoroughly and transfer 18 µL from each of wells A1

the example below. Regardless of the detection chemistry used in Optimizing qPCR through E5 to wells A8 through E12. the final assay, the best assay sensitivity will be obtained if primer concentrations are optimized in the presence of SYBR Green. This 5. Add 2 µL template-containing DNA (10-50 ng genomic DNA or allows detection of primer-dimer and other non-specific products, 0.1-1 ng plasmid) or RNA (10-100 ng total RNA or 0.5-10 ng and helps the user to screen out reactions with multiple products mRNA) to one set of reactions (columns 1-5) and 2 µL water to (step 8, next section). Alternatively, if maximum sensitivity is not the other (columns 8-12). a concern, the corresponding probe may be included in reactions 6. Perform thermal cycling: at 250 nM. Time Time Primer Optimization Example Number Temperature for for of Cycles 1. Prepare and dispense diluted primers qPCR qRT-PCR a. Prepare 60 μL of 8 μM working solutions of both forward Reverse (fwd) and reverse (rev) primers in the first tubes of 2 1 45 °C 0 min 15-30 min transcription separate 8-tube strips. b. Dispense 30 μL of water into tubes 2-5. Denature 1 94 °C 3 min 3 min c. Transfer 30 μL of the 8 μM primer solution from tube 1 into Denature 40 94 °C 15 sec 15 sec tube 2. Mix thoroughly by pipetting up and down at least Anneal, extend, 5 times. and read 60 °C 1 min 1 min d. Repeat transfer and mixing from tube 2 to 3, 3 to 4, and fluorescence 4 to 5. Dissociation/ e. Using a multichannel pipettor, transfer 5 μL from the strip- 1 * * * tubes containing diluted fwd primer into the first 5 wells melting curve down columns 1-5 of a 96-well PCR plate. After adding *See manufacturer’s instructions for the real-time thermal cycler used. reverse primer, PCR mix, and template (below), final 7. Evaluate fluorescence plots D( Rn) for reactions containing concentrations of forward primer will be 1000, 500, 250, target nucleic acid (columns 1-5). Primer combinations with the 125, and 62.5 nM. lowest Ct and the highest fluorescence will give the most f. Similarly, transfer 5 μL from the strip-tubes containing diluted sensitive and reproducible assays. rev primer into the first 5 wells across rows A-E. After adding PCR mix and template (below), final concentrations of 8. Evaluate dissociation/melting curves. Primer combinations reverse primer will be 1000, 500, 250, 125, and 62.5 nM. with single, sharp peaks in the presence of target nucleic acid (columns 1-5) and nothing detected in the corresponding 2. Prepare qPCR or qRT-PCR master mix (for 52 × 20 µL reactions): no-template control (columns 8-12) will give the most sensitive qPCR and reproducible assays. If all primer combinations give some product in the absence of template, and this no-template Reagent Catalog Number Volume product melts at a lower temperature than that with template, Water W1754 155 µL select the combination that gives the least amount of lower- SYBR Green JumpStart™ Taq melting no-template product. The latter is likely primer-dimer. S9939* 520 µL ReadyMix™ Detection can be avoided, or at least minimized, by adding a 15 second melting step approximately 3 °C below the melting Reference dye** R4526* 1.0 µL temperature of the desired PCR product during which fluores- qRT-PCR cence is measured after the annealing/extension step in each cycle. Reagent Catalog Number Volume Water W1754 123.8 µL Optimize Probe Concentration For maximum sensitivity, 250 nM probe may be used in all assays. SYBR Green JumpStart™ Taq S9939* 520 µL However, if maximum sensitivity is not required, lower levels of ReadyMix™ probe may suffice, thereby reducing the assay cost. To optimize Reference dye** R4526* 1.0 µL probe concentration, test the probe at several levels from 50 to 40 U/µL RNase inhibitor R2520 26 µL 250 nM final concentrations in PCR with optimized levels of 200 U/µL MMLV reverse primers and the lowest level of target nucleic acid expected. The M1427 5.2 µL transcriptase lowest level of probe that allows acceptable detection (Ct ≤ 30 for best reproducibility) may be used. * S9939 and R4526 are components of Catalog Number S4438. ** Use 10 × more for ABI 7700; replace with FITC for BioRad iCycler.

Our Innovation, Your Research — Shaping the Future of Life Science 19 20

Optimizing qPCR or design alternative primers. or designalternative efficient. To improve efficiency, optimizeprimerconcentrations levels oftargetinputmaynotbeaccurateifthereaction isnot –3.0 (80-110%efficiency)isgenerallyacceptable.Calculated efficiency from theslopeoflineusingequation: level andperformalinearregression. Calculatethereaction available, prepare aplotofCtversusthelognucleicacidinput user guidefortheinstrumentbeingused).Ifthisfeature isnot to prepare astandard curveandtocalculateefficiency(seethe software formostreal-time qPCRinstrumentscanbesetup also includeameltcurvetestattheendofthermo­ SYBRGreenprobe dye-baseddetection, concentrations.With with ano-templatecontrol, usingpreviously optimizedprimerand expected intestsamples.ConductqPCRwithalldilutionsand series toextendpastboththehighestandlowestlevelsoftarget or RNAsamplethatcontainsthePCRtarget.Planthisdilution preferably fiveormore, 4-to10-foldserialdilutionsfrom aDNA precision, sensitivity, andworkingrange.Prepare atleastthree, but dilution oftemplate,isanexcellenttooltotestassayefficiency, A standard curve,generatedbyperformingqPCRwithaserial Validate PerformancewithaStandard Curve Optimizing qPCR the assay. IfR line, andisinfluencedbypipettingaccuracytherangeof well thedatafitsmodelandhowonastraight be –3.33(100=100%10 double witheachcycleandtheslopeofstandard curvewill If thePCRis100%efficient,amountofproduct will pipettor properly andthatthevolumedispensedisreproducible. accuracy maybeaproblem. Verify thatthepipettetipsfit if severalrandompointsare aboveorbelowtheline,pipetting situation, addlessordilutethesamplenucleicacid.Alternatively, target exceedstheusefulassayrange(Fig.11B).To address this it islikelythatthereaction issaturatedandthatthelevelof nucleic acidare shiftedawayfrom thelinearregion oftheplot, Similarly, ifoneormore pointsatthehighestlevelsofinput optimize primerconcentrationsordesigndifferent primers. level exceedsassaysensitivity(Fig.11A).To improve sensitivity, shifted awayfrom thelinearregion oftheplot,itislikely thatthe If oneormore pointsatthelowestlevelsofinputnucleicacidare The correlation coefficientoftheline,R sigma.com 2 is≤0.985,theassaymaynotgivereliable results. Efficiency =10 ORDER: 800-325-3010 (–1/–3.33) -1). Aslopebetween–3.9and (–1/slope) 2 , isameasure ofhow -1 TECHNICAL SERVICE: 800-325-5832 cycling. The

high levelsofinputnucleicacid. A. Assaynotlinearatlowlevelsofinputnucleicacid.B. A. B. qPCR Optimization Figure 11.UseofStandard CurvestoEvaluate Ct Ct 10 15 20 25 30 35 24 26 28 30 32 34 36 38 40 5 -5 -3 -4 Log ofDNAdilution Log ofDNAdilution -2 -3 -2 -1 -1 0 0 INNOVATION @ WORK

Prepare a Melt Curve Setting the Threshold Value Since SYBR Green binding dye is a non-specific dye that will detect There are several methods that can be used to calculate the any double-stranded DNA, it is important to verify that the PCR threshold value. The critical factors for determining the level of produces only the desired product. This can often be detected when the threshold are: (a) the fluorescence value must be statistically Optimizing qPCR PCR efficiencies are larger than 120%. Melt, or dissociation, curve higher than the background signal, (b) the samples must all be analysis can also be used to determine the number and approxi- measured in the exponential phase of amplification and (c) the mate size of products. An assay with high specificity will give efficiency of amplification must be identical for all samples. a single peak at a high temperature (> 80 °C) in all reactions The fluorescence value must be statistically higher than the and nothing, or very little, detected in the no-template controls background signal to ensure that real data are collected. Most (Fig. 12A). If the melting curve has more than one major peak, instruments automatically calculate a threshold level of fluores- as in Figures 12B and 12C, the identities of the products should cence signal by determining the baseline (background) average be determined by fractionating them on an ethidum bromide- signal and setting a threshold 10-fold higher than the baseline stained agarose gel. As shown in Figures 12E and 12F, reactions B average signal. and C contain excessive amounts of primer-dimer or other non- specific products. Lowering the primer concentrations will often Setting a manual threshold is best accomplished using a log reduce the amount of non-specific products. If non-specific signal plot, as the exponential part of the curve shows clearly products are still detected in significant amounts with low primer as a linear trace. levels, redesign the primers.

Figure 12. Evaluation of Melt Curves A. B. C.

D. E. F.

Melt, or dissociation, curves showing a sharp peak of specific product at > 80°C, very little non-specific product at lower temperatures (A), or significant amounts of non-specific, lower melting product (B&C). D-F show PCR products from A-C, respectively, fractionated on ethidium bromide-stained 2% agarose gels.

Our Innovation, Your Research — Shaping the Future of Life Science 21 22

Optimizing qPCR Optimize your Quantitative Real-Time RT-PCROptimize yourQuantitativeReal-Time (qRT-PCR). For additionalinformation,pleaseseeAppendix2:Howto Nanochip andintegritywasevaluatedbefore useinqRT-PCR. Samples oftotalRNApreparations (~150ng)were fractionatedonaRNA6000 sample 11(RIN=9.8)thanin97.0). correlate toRIN,asmRNAs were detectedupto2cycleslaterin with Oligo-dTtoprimeRT. Thelatterdifference wasshownto mRNA amounts varying by four-fold when using two-step qRT-PCR primers. ThissameRNAtemplatewasquantifiedtohave relative mRNAs whenusingaone-stepqRT-PCR usinggene-specific shown inFigure 13,gavequantifiabledifference forseveralrare By wayofexample,theRNAsampleswithBioanalyzertraces qRT-PCR successmustbedeterminedempiricallyforeachassay. abundant mRNA.Therefore, thecorrelation betweenRINand integrity willberequired todetectarare mRNAcompared toan one-step RT-PCR withgene-specificprimers.Inaddition,higher will require muchhigherintegritythanastrategythat uses transcription andPCRprimersnearthe5’-endofalongcDNA RT-PCR strategythatusesatwo-stepOligo-dTtoprimereverse sensitivity required aswelltheRT-PCR strategy. Forexample,an will givesatisfactoryresults inqRT-PCR dependsuponthelevelof has aRINof1.WhetherornotpartiallydegradedRNA(RIN<9) intact RNAhasaRINof10whereas completelydegradedRNA integrity number(RIN)fortheRNAsampleanalyzed.Perfectly between, andaftertherRNApeakstodeterminearelative software evaluatestheproportion ofRNAdetectedbefore, electrophoresis withanAgilent2100bioanalyzer. Theinstrument The qualityoftotalRNAismostreadily assessedbycapillary Verify RNAQuality n n n n results, thefollowingpointsshouldbeaddressed aswell: the guidelinesforstandard qPCR,butforoptimumqRT-PCR When performingqRT-PCR, itisnotonlyimportanttoconsider PCR (qRT-PCR) Quantitative ReverseTranscription Additional Guidelinesfor Optimizing qPCR

Verify RNAquality Optimize reverse transcription Conduct no-reverse controls transcriptase(no-RT) Confirm thatprimersspanorflanklongintrons Electropherogram Agilent Bioanalyzer2100 Figure 13.Evaluationof RNA Integritywiththe sigma.com ORDER: 800-325-3010 RIN =7.0 sample 11 RIN =9.8 sample 9

TECHNICAL SERVICE: 800-325-5832 Virtual Gel Virtual

lanes 3and4ofFigure 14are partiallydegraded. two rRNAbands,asinlanes1and2Figure 14.TheRNAin The mRNAshouldappearasalightsmear, mostlybetweenthe band shouldhaveapproximately twicetheintensityoflower. discrete bandsatapproximately 5kband2theupper quality totalRNA,thetwolargestrRNAsshouldappearas sis toverifythattheRNAappearsreasonably intact.Forgood evaluated byethidiumbromide-stained agarose gelelectrophore- If aBioanalyzerisnotavailable,1-2µgoftotalRNAmaybe determine whetherornotdigestion withDNaseIisneeded. free controls oramplification-gradeDNaseI.Conductno-RT to introns, itmaybenecessarytodigestinputRNAwithanRNase- unknown, orifthere are nosuitableprimersthatspanorflank If thegeneofinterest hasnointrons, iftheintron positionsare multiple smallintrons. exon-exon junctions,flankalong(severalkb)intron, orflank figures illustratesthat,ifpossible, primersshouldeitherspan RT-PCR.likely willbeamplifiedduring Theexampleinthesetwo the genomicDNA,whileallotherintrons are short(<1kb)and Figure 16islongenough(~ 6.5kb)topreclude amplificationof RT-PCRbe amplifiedin (e.g.Fig.17A).Forexample,Intron 1in DNA sequenceswithshortintervening(~1kb)may against thegenomicdatabasefortargetorganism(Fig.16). identified byperformingaBLASTsearch withthecDNAsequence for thetargetmRNAare publicly available,intron positionscanbe exon-exon junction(Fig.15).IfbothgenomicandcDNAsequences intron thatisnotpresent inthemRNAsequenceorthat spanan RT-PCR,amplification during useprimersthateitherflankan lead toerroneously highestimatesofmRNAlevels.To avoidDNA target mRNAislessthan100copies/cell,DNAamplificationcan in comparisontotheproducts from theRNA.If,however, the thousands ofcopiespercell),DNAamplificationwillbenegligible as wellRNA.IfthetargetmRNAisfairlyabundant(hundreds or single moleculeofDNA,RT-PCR canamplifycontaminatingDNA removes 100%oftheDNA.BecausePCRcanamplifyevena While most DNA is eliminated during RNA purification, no procedureConfirm thatPrimersSpanorFlankLongIntrons buffer andstainedwithethidium bromide. Samples oftotalRNA(2μg)were fractionatedona1% agarose gelinTBE Gel Analysis Figure 14.EvaluationofRNAIntegritybyAgarose 28S rRNA 18S rRNA 12 3 4

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Figure 15. Illustration of Intron-Spanning (A) and Figure 17. Evaluation of No-RT Controls Intron-Flanking (B) Primers for RT-PCR A. B. A. Primers Span Intron mw + –+ – +/– RT

P2 Optimizing qPCR P2 DNA: P1 P1 2,000 1,500 P2

mRNA: P1 1,000

B. Primers Flank Intron 750 P4 DNA: 500 P3 300 Introns are in red, exons are in green. Primers P1 & 2 span an intron and primers 294 P3 & 4 flank an intron. Note that primers P1 & 2 are only partly complimentary 150 191 to the gDNA strand and will not generate a PCR product from DNA unless the 50 annealing temperature is extremely low. P3 & 4 may generate a longer PCR product from DNA if the intron is short (~1 kb), but not if it is sufficiently long (several kb). RT-PCR products produced in the presence (+) or absence (-) of RT enzyme were fractionated on an ethidium bromide-stained 2% agarose gel in TBE. Figure 16. BLAST Alignment of cDNA Sequence with Primers for the mRNA target in (A) flank a 1 kb intron. Note the 1.5 kb band Genomic DNA Sequence in the no RT control. The mRNA target in (B) aligns with several genes, at least one of which is a pseudogene that lacks the intron between the primers used for RT-PCR. As such, the no RT control gives a larger yield than when reverse transcriptase is added.

Conduct No-Reverse Transcriptase (No-RT) Controls Regardless of whether primers span or flank introns, the specificity of qRT-PCR assays should be tested in reactions without reverse transcriptase (no-RT control) to evaluate the specificity of DNA amplification. As mentioned above, DNA sequences with short introns (≤ 1 kb) may be amplified in RT-PCR. Many genes have additional copies, or pseudogenes, that lack one or more introns (Fig. 17B). As a result, qRT-PCR assays should be tested for potential DNA-only amplicons by performing reactions that contain RT, the same RNA, but no RT enzyme. DNA amplification is not a problem if the Ct values for no-RT reactions are at least 5 cycles greater (32-fold less) than those for reactions with RT. However, if there are fewer than 5 cycles between Ct values for reactions with and without RT, DNA amplification may skew The complete cDNA sequence for rat p53 from Genbank (accession number attempts at mRNA quantitation. NM_030989) was used in a megaBLAST search for identical sequences in the rat genome (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html). The alignment In cases where DNA amplicons contribute significantly, one should on chromosome 10 is shown; each tick mark on the scale represents 1 kb. digest the RNA with an RNase-free or amplification grade DNase I before qRT-PCR to allow reliable mRNA quantitation. Note that on-column DNase digestion, which is commercially available in several RNA purification kits, is less effective at eliminating DNA than digestion in solution after eluting RNA from the column. As a result, on-column (OC) DNase digestion may or may not be sufficient for qRT-PCR (Fig. 18, see +OC DNAse -RT samples versus post prep DNAse -RT). No-RT controls should be conducted with DNase-digested RNA to verify that the digestion was successful and sufficient. In the example shown in Figure 17, OC DNase digestion is sufficient to reliably detect the target mRNA. It would not be sufficient to reliably quantitate a less abundant mRNA, if the samples contained less of the mRNA shown, or if greater sensitivity was required.

Our Innovation, Your Research — Shaping the Future of Life Science 23 24

Optimizing qPCR and randomoligomers willgivethebestresults. or thepolyadenylationlevelofRNAs varies,amixture ofOligo-dT decamers willgivebetterdetection. IfthelocationofqPCRtargets polyadenylated, randomhexamers, octamers,nonamers,or more thanafewkilobasesfrom the3’-endorifRNAisnot choice forprimer. Ontheotherhand,ifqPCRtargetsare are nearthe3’-endofpolyadenylatedmRNAs,oligo-dTisa good performed withaliquotsfrom thecDNApool.IfallqPCRtargets This wouldbefollowedbyseparateqPCRassaysforeachtarget non-specific primersmaybeusedtogenerateapoolofcDNA. thatcanoccurwithgene-specificprimers, variations inRT carefully considered. To avoidthepotentiallyhighinter-assay primingshouldbe Give thesecomplications,thechoiceofRT low abundance RTmRNAs primers. not detected using non-specific encode thegeneofinterest andmayallowquantitationofvery hand, withagene-specificprimer,RT product allofthe will complicating comparisonsbetweenRNAlevels.Ontheother separate reactions mayhave verydifferent efficiencies,thus primers require separatereactions foreachtargetRNA.These nonamers, decamers,ordodecamersmaybeused.Gene-specific assay, areverse gene-specificprimer, Oligo-dT, randomhexamers, specific primersmustbeused.Whenperformingatwo-step greatly affect qRT-PCR results. Forone-stepqRT-PCR, gene- The choiceofprimersusedtoinitiatereverse transcriptioncan Optimize ReverseTranscription Sigma GenElute Total RNAwasprepared from 30mgpiecesofmouseliverwitheitherthe new startingmaterialandRNApreparation method. should be with performed and at without least RT once with each give different levelsofcontaminatingDNA.Asaresult, reactions specific mRNAs.Inaddition,different RNApurificationmethods growth conditions,produce significantlydifferent levelsof Note thatdifferent typesofcellsandtissues,aswelldifferent Optimizing qPCR are shown.Similarresults were obtainedwithbothmanufacturers’ products. were usedinone-stepqRT-PCR. Fluorescence plotsfortwooftheRNAsamples DNase Iaccording tothemanufacturer’s instructions.Equalproportions ofall without on-columnDNasewere digestedwithSigma’s AmplificationGrade DNase digestion.Afterpurification,aliquotsofthefourRNAsamplesprepared tive manufacturer’s on-columnDNaseproduct andtwowere prepared without manufacturers’ instructions. Two RNAsampleswere prepared withtherespec - (OC) withPost-Preparation DNaseDigestion Figure 18.ComparisonofOn-ColumnDNaseDigestion Fluorescence (dRn) 0 1 2 3 4 5 24 sigma.com 68 ™ Total RNAKitortheQiagenRNeasyMiniaccording tothe 10 12 14 16 ORDER: 800-325-3010 18 + RT +/– DNase 20 Cycles 22  24 26 28 30  – RT – DNase 32 34 TECHNICAL SERVICE: 800-325-5832 – RT + post-prepDNase 36 – RT + OCDNase 38 40 42 44

20 units(Fig.19B).Superscript MMLV-RT (Fig.19C)gavebetterspecificitythan reactions with results. AsshowninFigure 18,one-stepreactions with2unitsof enzymeperreactionThe amountofRT canalsoaffect qRT-PCR gene specificprimersthatcanforma3’-duplex(Fig.19B). give lessprimer-dimer (Fig.19D)thanone-stepqRT-PCR with RT andhot-startTaqspecific primerfor polymeraseforqPCRmay (Fig. 19A).Similarly, performingtwo-stepRT-PCR withanon- (Fig. 19B)thanwhenthereaction wasperformedat37°C Transcriptase)(Moloney MurineLeukemiaVirus-Reverse at45°C product inone-stepqRT-PCR wasperformedwithMMLV-RT whenRT the primersusedinFigure 18gavesignificantlylessnon-specific enzyme mayreduce theamountofprimer-dimer. Forexample, at whichtheenzymeisfullyactiveorusingahigh-temperature Increasing incubationtemperature RT tothehighesttemperature template, primer-dimer step. formationmaystartduringtheRT tures. enzymescanextendaDNAprimeron SinceRT strong 3’-duplexwillhybridizemore readily atlowertempera- especially withgene-specificprimers.Primersthatcanforma The temperature reactions usedforRT mayaffect specificity, be limitedtonomore than10%ofthefinal reaction volume. Taq activity, product theamountofRT transferred toqPCRshould two-step reactions, enzymecaninterfere butbecausetheRT with ture reaction. RT maygivebetterresults HigherlevelsofRT in present toformnon-specificproducts duringthelow tempera- attributed tothefactthatgene-specificprimersare not RT-PCRspecificity thandoesone-step (Fig.19D).Thiscouldbe oftengivesgreaterwith Oligo-dTorrandomprimersforRT those showninFigure 18(datanotshown).Two-step RT-PCR MMLV-RT, andOmniscriptfrom Qiagengaveresults similarto ™ III,anRNaseH – deletionof

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Figure 19. Optimization of RT A. B. Optimizing qPCR

C. D.

Melt curves of RT-PCR products produced with one-step (A-C) or two-step (D) qRT-PCR. Reactions (A-C) each contained 10 µL of SYBR Green JumpStart Taq ReadyMix, 0.02 µL of Reference Dye, both gene-specific primers at 0.4 µM, and 10 ng human total RNA in a final volume of 20 µL. Gene-specific primers were 5’-CGGGCTTCAACGCAGACTA-3’ and 5’-CTGGTCGAGATGGCAGTGA-3’ for c-fos (Accession NM_005252). Reactions (A&B) also contained 20 units of MMLV- RT, whereas reaction (C) contained 2 units. Reaction A was incubated at 37 °C for 30 min before qPCR, whereas (B&C) were incubated at 45 °C for 30 min before qPCR. In (D), the RT reaction contained 1× MMLV buffer, 0.5 mM dNTPs, 1 µM Oligo-dT, 0.8 units/µL RNase inhibitor, 200 units MMLV-RT, and 10 ng human total RNA in a final volume of 20 µL. The reaction was incubated at 25 °C for 10 min, 37 °C for 50 min, and 80 °C for 10 min. 2 µL of the RT reaction product was added to qPCR containing 10 µL of SYBR Green JumpStart Taq ReadyMix, 0.02 µL of Reference Dye, and both gene-specific primers at 0.4 µM as for the one-step reactions (A-C). All qPCR reactions were incubated at 94 °C for 3 min to denature, then for 40 cycles of 94 °C for 15 sec and 60 °C for 1 min.

Our Innovation, Your Research — Shaping the Future of Life Science 25 26

Optimizing qPCR high Ctvalues,withintherangeof50-500nM. low Ctvaluesand/orincrease concentrationsforthosethatgive Decrease primerconcentrations forthoseprimerpairsthatgive improve results ifsensitivityisunacceptableinmultiplexreactions. On theotherhand,optimizingprimerconcentrationswilllikely primer levelsifmultiplexandsingleplexreactions givesimilarresults. with allprimerscombined(multiplex).There isnoneedtomodify of targetsexpectedforeachprimerpairalone(singleplex)and will bebeneficial,prepare standard curvesthatcovertherange amplification ofalltargets. To determineifsuchadjustments Adjusting thelevelsofprimersmayallowamore balanced reaction, usingupreactants before othertargetsare detectable. than theother(s),amplificationofthattargetmaydominate higher D than theother(s)orifoneprimerpairgivesamuchlowerCt If onetargetinamultiplexreaction issignificantlymore abundant Optimize PrimerConcentrations and efficiency. the primerscross-react orotherwisealterreaction specificity individual primersworksingly, butwhencombined inmultiplex several orallprimercombinations.Itisveryoftenthecasethat optimizations shouldbeperformedaswelloptimizationwith Primer DesignforPotentialonpage18.Individualreaction (DG ≥–2.0kcal).Formore information,seethesectionCheck (Tm difference ≤2°C)andnonecanformstrong 3’-duplexes if allprimersinthereaction havesimilarmeltingtemperatures As forsingle-targetreactions, multiplexqPCRwillgivethebestresults Check PrimerDesign n n n optimize thefollowing: fluorophores. Formultiplex reactions, itisalso recommended to contains Traits ofCommonFluorophores toaidintheselectionof bandwidths are usefulformultiplexapplications.Appendix1 analysis. Asaresult, fluorophores withnarrow, well-resolved of themultipleemissions.Thisfacilitatessignalisolationanddata tion. Onesimpleconsiderationistominimizethespectralseparation quantitated inasinglereaction, oftenrequire additionaloptimiza- Successful multiplexqPCR,inwhichmore thanonetargetis Multiplex Reactions Additional Optimizationfor Optimizing qPCR

Optimize Mg Optimize primerconcentartions Check primerdesign R (theamountoffluorescence intheno-templatecontrol) sigma.com 2+ concentration ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

and efficiencyofthelater reaction. running thereactions togetherdramaticallychangesthesensitivity give relatively similarefficienciesandsensitivities(y-axisvalues) that whiletheindividualreactions (darkblueandgreen lines) performed individuallyandtheninmultiplex.Thegraphshows this point. The efficiency curves for two primer/probe targets were creating majorchangesinPCRefficiency. Figure 20demonstrates competition forreagents andexacerbatesanynon-optimalconditions multiplex PCR.Runningmultiplereactions concurrently introduces These effects are magnifiedwhenoneattemptstoperform and product Tmchangeasthecationconcentrationincreases. PCR requires aminimalamountofmagnesium,andbothefficiency less drasticforthesemonovalentcations. such asKCl,willalsochangeDNAduplexTm,buttheeffect is effect creating sideproducts andsappingPCRefficiency. Salts, DNA duplexesbecomesubstratesforthepolymerase,in priming eventsandprimer-primer interactions.Themis-primed increase theaffinityofprimerstoward hybridization,includingmis- ture, ofaDNAduplex.Itfollowsthathighmagnesiumlevels and increasing magnesiumraisesthestability, ormeltingtempera- Divalent cationsstrongly affect DNAdouble-strandhybridization, cationic counter-ion fordNTPsandaco-factorallpolymerases. Magnesium playsseveralroles inPCR.Itisarequired divalent Optimize Mg 2+ Concentration

INNOVATION @ WORK

Figure 20. Singleplex Reaction vs. Duplex Reaction

&0<& IDP VLQJOH &0<& IDP GXSOH[ Optimizing qPCR 6LQJOHSOH[YVGXSOH[ &'& -2( VLQJOH  &'& -2( GXSOH[ /LQHDU &0<& IDP VLQJOH /LQHDU &0<& IDP GXSOH[ /LQHDU &'& -2( VLQJOH /LQHDU &'& -2( GXSOH[



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FP\FGXSOH[ \ [ 5  

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        ORJQJ'1$

Our Innovation, Your Research — Shaping the Future of Life Science 27 28

qPCR Reagent Selection Table Package sizesbasedon50µLreactions qPCR ReagentSelectionTable the reagent toSYBRGreen dyeorprobe-based formulations. capillary-based instruments.Thetableisread verticallytonarrow cycler format.Products are compatiblewithtube,plate,or for yourneeds.Thetableisread horizontallytoselectthethermal Use thefollowingtabletochoosemostappropriate reagent qPCR ReagentSelectionTable Probe based qPCR SYBR Green based qPCR S9194 Number Catalog QR0100 D7440 S4438 Number Catalog Plate/Tube Instruments QR0200 D6442 D9191 S5193 sigma.com reference dye) Quantitative PCR(withinternal ReadyMix forHigh-Throughput SYBR Green JumpStartTaq Product Name RT-PCR Kit SYBR Green Quantitative Quantitative PCR JumpStart Taq ReadyMixfor ReadyMix forQuantitativePCR SYBR Green JumpStartTaq Product Name Quantitive RT-PCR ReadyMix reference dye) PCR (withinternal High-Throughput Quantitative JumpStart Taq ReadyMixfor with dUTP JumpStart Taq ReadyMix ReadyMix withoutMgCl SYBR Green JumpStartTaq (with separatetubeofMgCl ORDER: 800-325-3010 2

2 ) TECHNICAL SERVICE: 800-325-5832 Package Size (100 Reactions) 1 Kit 2000 Reactions 400 Reactions 20 Reactions (100 Reactions) 1 Kit 2000 Reactions 400 Reactions 20 Reactions 400 Reactions 100 Reactions 20 Reactions 400 Reactions 100 Reactions 500 Reactions 100 Reactions Package Size 400 Reactions 100 Reactions 20Reactions as thestartingtemplate. reagents are notedingray. Theseare forusewhenRNAisused to eliminateanextrapipettingstep.TheQuantitativeRT-PCR High-throughput mixesincludethereference dyeintheReadyMix Number Catalog QR0100 QR0200 D9191 S1816 Number Catalog Glass CapillaryInstruments S5193 (with separatetubeofMgCl Product Name RT-PCR Kit SYBR Green Quantitative Quantitive RT-PCR ReadyMix with dUTP JumpStart Taq ReadyMix Capillary Formulation ReadyMix forQuantitativePCR, SYBR Green JumpStartTaq Product Name ReadyMix withoutMgCl SYBR Green JumpStartTaq 2

2 ) 400 Reactions 100 Reactions 20 Reactions Package Size (100 Reactions) 1 Kit (100 Reactions) 1 Kit 400 Reactions 100 Reactions 20 Reactions 400 Reactions 100 Reactions 20 Reactions Package Size INNOVATION @ WORK

Troubleshooting

Fluorescence Issues No, or low, fluorescence in both the test sample and in the positive control with the correct PCR Number 1 product on the gel Troubleshooting SYBR Green Dye-Based Detection Probe Detection Possible cause Bad SYBR Green binding dye High-background fluorescence Degraded probe Compare fluorescence of the SYBR Check the raw fluorescence Digest 5 fmoles of probe with Green binding dye mix ± 1 µg DNA. (multicomponent plot). Fluorescence DNase I. Fluorescence should be at Diagnostic test Fluorescence should be at least should increase at least 10,000 units least 10,000 units greater than 10,000 units higher with DNA between cycles 1 & 40 without DNase digestion added than without Purchase new SYBR Green binding Purchase a new probe Purchase a new probe Solution dye or a new qPCR mix with SYBR Green binding dye Number 2 Low fluorescence from test sample, but the positive control has good fluorescence Possible cause Fluorescence quenching Diagnostic test Positive control gives good fluorescence Solution Purify the input nucleic acid Number 3 Declining or hooked fluorescence plots As PCR product accumulates, the complimentary strand competes with the primer and/or probe for annealing Possible cause to template Diagnostic test See figure Solution Ignore it if the Ct is not affected

Figure 21. Hooked Fluorescence Plots

Our Innovation, Your Research — Shaping the Future of Life Science 29 30

Troubleshooting Troubleshooting Solution Diagnostic test Possible cause Number 8 Possible cause Number 6 Solution Diagnostic test Possible cause Number 5 Solution Diagnostic test Possible cause Number 4 Solution Diagnostic test Possible cause Number 7 Solution Diagnostic test sigma.com product isspecific.Continue tousetheprimers This isnotaproblem ifthe gelanalysisshowsthatall or repeats Check sequenceofampliconforAT orGC-richregions PCR product Localized AT or GC-richregions orshortrepeats in Single product ongel Multiple Tmpeaks The Mg There are multiplepeaksinthedissociationplot/melt curve Try addingBSAto0.3%inthePCRorpurifyinputnucleicacid amplification oftheexogenoustarget Test amplificationwithadilutedsample.Spikethe reaction withalowlevelofexogenoustargetandtestfor Inhibition inthetestsampleislikely No amplification results from asampleknowntocontaintargetandthepositivecontrol doesamplify Purchase anewreference dye.Alwaysprotect dyefrom lightduringstorage plots shouldbecomesmoothiffluorescence isnotnormalizedtoaninactive reference dye instead ofDRn(thedifference ofthereporter fluorescence inthesampleandthatnotemplatecontrol). The Disable reference dyenormalizationorlookattheresults forDR(leveloffluorescence intheno-templatecontrol) Bad reference dye The fluorescence plotssuddenlyspikeupwards optimum annealingtemperature incubation temperature step fortheRT Try RT-PCR, lowerprimerconcentrations.With enzymeandusea2-step and/orahigher tryusinglessRT will appearasadiffuse bandat≤50bp Fractionate thePCRproduct onanethidiumbromide-stained agarose gelorusetheBioanalyzer. Theprimer-dimer Primer-dimer, generatedfrom primersthatannealattheir3’-ends,extend,andthenamplify There isabroad peakatalowerTmthanthedesired product, especiallyinloworno-templatereactions Titrate theMg Titrate multiple products Fractionate thePCRproduct onanethidiumbromide-stained agarose gelorusetheBioanalyzertoverify ORDER: 800-325-3010 2+ concentrationinthereaction istoohighortheannealingtemperature istoolow 2+ todeterminetheoptimumconcentration.Performannealingtemperature gradienttoselectthe TECHNICAL SERVICE: 800-325-5832 Dissociation/Melting Curves Fluorescence Issues Design uniqueprimers source organismtoverifysingletarget BLAST primersequencesagainstthesequenceof Multiple targetsinthesource material Multiple products ongel INNOVATION @ WORK

Standard Curve Number 9 PCR efficiency < 80%

Suboptimal PCR conditions (See example. This illustrates Poorly designed primers Troubleshooting Possible cause low Taq activity) Prepare or purchase fresh PCR mix Check the primer design. Test the PCR mix with a set Diagnostic test of primers known to work well and a positive control template Solution Prepare or purchase fresh PCR mix Design new primers Number 10 PCR efficiency is greater than 120% Possible cause Excessive primer-dimer Pipetting is inaccurate Inhibition Evaluate dissociation plot as shown Test pipette calibration See Problem Number 4: The Diagnostic test in the figure below fluorescence plots suddenly spike upward Try lower primer concentrations. If the pipettes are inaccurate, get See Problem #4: The fluorescence With RT-PCR, try using less RT them re-calibrated. If the pipettes plots suddenly spike upward enzyme, doing 2-step and/or using are properly calibrated, practice Solution a higher incubation temperature for pipetting accurately the RT step

Our Innovation, Your Research — Shaping the Future of Life Science 31 32

Troubleshooting Troubleshooting Solution Diagnostic test Possible cause Number 12 Solution Diagnostic test Possible cause Number 11 Figure 23.Standard CurvethatisnotLinearwith HighLevelsofSampleInput Figure 22.Standard CurvethatisnotLinearwithLowLevelsofSampleInput sigma.com Use lessinputnucleicacid Low Ct(<12-15,dependingoninstrument) Exceeded assaycapacity The standard curveisnotlinearwithhighlevelsofsampleinput Optimize primerconcentrations Ct >30;qPCRreplicates highlyvariable Exceeded assaysensitivity The standard curveisnotlinearwithlowlevelsofsampleinput ORDER: 800-325-3010

Ct Ct 10 15 20 25 30 35 5 24 26 28 30 32 34 36 38 40 -5 -3 TECHNICAL SERVICE: 800-325-5832 -4 Standard Curve -2 Log ofDNA Log ofDNAdilution -3 dilution -2 -1 -1 0 0 INNOVATION @ WORK

qRT-PCR Specific Number 13 Product is detected in the no-RT reaction with primers that flank an intron

The intron is short enough that the primers amplify Pseudogenes are present that lack introns Troubleshooting Possible cause (~1.4 kb in example) Fractionate the PCR product on an ethidium bromide- Fractionate PCR product on ethidium bromide-stained Diagnostic test stained agarose gel. No-RT product will migrate more agarose gel. No-RT product will migrate the same as slowly than +RT product as in gel photograph shown +RT on gel Solution Re-design the primers to flank or span a longer intron or multiple introns

Multiplex Number 14 One, or more, primer/probe set does not work well in a multiplex reaction Possible cause The target is less abundant or the primers are less efficient Diagnostic test Conduct reactions with individual primer/probe sets to see if they work adequately in the absence of competition Limit the level of primers that dominate the reaction and give higher signal. Determine the level that reduces DRn Solution without increasing Ct significantly

Sigma Product Specific Number 15 Sigma’s JumpStart Taq doesn’t work as well as Qiagen’s HotStarTaq Possible cause Reactions were denatured at 95 °C for 15 min before cycling Diagnostic test Check the thermocycling profile Solution 3 min at 94 °C is sufficient to activate JumpStart Taq; longer incubations will partially inactivate Number 16 Sigma’s ReadyMix doesn’t work, but competitors do Possible cause REDTaq ReadyMix was used for real-time qPCR Diagnostic test Check color of qPCR mix Solution Use clear mixes for real-time qPCR. Red dye will interfere with fluorescence detection for most instruments

Our Innovation, Your Research — Shaping the Future of Life Science 33 34

Appendix 1 Traits ofCommonlyUsedFluorophores Appendix 1 6-FAM Fluorescein Erythrosin BODIPY Oregon Green Eosin AMCA NBD LightCycler Cascade Blue BODIPY BODIPY BODIPY BODIPY BODIPY BODIPY BODIPY BODIPY BODIPY EDANS DABCYL Cy Cy Cy Cy Cy NED Texas Red ROX TAMRA Rhodol Green Rhodamine Red Rhodamine Green LightCycler HEX JOE TET Cy Rhodamine 6G Oregon Green Oregon Green Acridin Yakima Yellow VIC ® ® ® ® ® ® ® ™ ™ 7 5.5 5 3.5 3 2 ™ ™ ™ ™ sigma.com ™ ® ® ® ® ® ® ® ® ® ® TMR 530/550 FL-Br2 650/665 630/650 TR 581/591 576/589 564/570 558/568 ™ ® ® 705 640 ® ™ Dye ® ® ® ™ ™ 514 500 488 ™ ™ ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832 Ab (nm) 492 529 521 353 466 335 453 362 685 625 544 534 531 646 625 588 581 575 563 558 743 675 643 581 552 489 535 520 521 494 396 526 538 546 583 585 565 496 560 504 524 506 499 492 max Extinction Coefficient (l mole not available not available 78,000 90,000 95,000 19,000 22,000 5,900 32,000 11,000 110,000 56,000 77,000 75,000 102,000 101,000 68,000 136,000 83,000 142,000 97,000 250,000 250,000 250,000 150,000 150,000 150,000 71,000 83,000 29,000 84,000 116,000 82,000 91,000 63,000 129,000 78,000 102,000 85,000 78,000 88,000 ­— ­— ­— –1 cm –1 ) Em None (nm) 520 553 544 442 535 493 462 705 640 570 554 545 660 640 616 591 588 569 569 767 694 667 596 570 506 556 548 536 518 410 448 603 605 580 523 580 532 550 526 519 517 554 575 max INNOVATION @ WORK

Appendix 2

How to Optimise Your Quantitative or in the first extraction buffer containing guanidine isocyanate, provided in the extraction kits, and others freeze it in liquid Real-Time RT-PCR (qRT-PCR) nitrogen. The extracted analyte (either total RNA or poly-

Michael W. Pfaffl, Physiology Weihenstephan, Technical University of Munich adenylated mRNA) as well as the RNA isolated using columns or a Appendix 2 (TUM), Weihenstephaner Berg 3, 85354 Freising Weihenstephan, Germany liquid-liquid extraction, could result in varying RNA qualities and http://optimisation.gene-quantification.info quantities. For example RNA extracted from collagen rich or adipose tissues often has a lower total RNA yield, is of lesser qual- Introduction ity, and contains partly degraded RNA sub-fractions. Particular RNA extraction techniques can work more effectively in one To get very reliable and quantitative real-time RT-PCR results, the specific tissue type compared with another one and result in up applied mRNA quantification assay and working procedure to 10-fold variations in total RNA yield per extracted tissue mass should be highly optimised. The efficacy of kinetic RT-PCR is and as a result, on the following real-time RT-PCR gene expres- measured by its specificity, low background fluorescence, steep sion analysis as well. A lot of total RNA preparations are contami- fluorescence increase and high amplification efficiency, and nated with genomic DNA fragments and protein at very low constant high level plateau. Therefore the typical reaction history levels. The enormous amplification power of kinetic PCR may st can be divided in four characteristic phases, Figure 1. The 1 result in even the smallest amount of DNA contamination phase is hidden under the background fluorescence, where an interfering with the desired specific RT-PCR product. To confirm nd exponential amplification is expected; 2 phase with exponential the absence of residual DNA, either a negative control (minus-RT rd amplification that can be detected and above the background; 3 or water) should be included in each experimental setup. It may phase with linear amplification efficiency and a steep increase of be necessary to treat the RNA sample with commercially available th fluorescence; and finally 4 phase, or plateau phase, defined as RNase-free DNase, to get rid of any DNA. However, unspecific the attenuation in the rate of exponential product accumulation, side reactions of the DNase often result in RNA degradation. normally seen in later cycles. However, it is always recommended to remove the DNase prior to any RT or PCR step. Furthermore, the design of the PCR product Figure 1 should incorporate at least one exon-exon splice junction to allow 60 a product obtained from the cDNA to be distinguished on electrophoresis from genomic DNA contamination. 50 4th phase RNA Quantity and Integrity Accurate quantification and quality assessment of the starting RNA 40 3rd phase sample is particularly important for an absolute quantification strategy that normalises specific mRNA expression levels against a 30 given calibration curve measured in molecules or concentrations/ grams RNA. The RNA quality assessment requires accurate quanti- 20 2nd phase fication of the isolated total RNA or mRNA fraction by optical background level density at 260 nm, (OD260), and determination of the RNA quality 10 1st phase calculated by the OD260/OD280 ratio or by the RiboGreen RNA Quantification Kit from Molecular Probes. Furthermore, the RNA 0 quality can be verified by capillary electrophoresis with the 010203040 Real-time PCR cycle Bioanalyzer 2100 on a microchip lab-on-chip system from Agilent Technologies. The recently developed RNA integrity number (RIN) The four characteristic phases of PCR. determines the level of intact total RNA on the basis of an electropherogram, Figure 2. The RIN value can range between The following information addresses optimisation strategies in 1-10: RIN 1 for totally degraded RNA, and RIN 10 for a perfect quantitative real-time RT-PCR. Special focus is laid on the pre- and intact total RNA. RIN numbers under 5 are at least partly analytical steps, sampling techniques, RNA extraction, and reverse degraded and result in low 18S rRNA and 28S rRNA peaks in the transcription (RT), primer usage, and post-analytical steps, especially electropherogram. In a broader sense, poor RNA quality and low on crossing point evaluation and crossing point measuring at RIN numbers influence the qRT-PCR performance significantly and elevated temperatures. lead to an inhibition of the PCR performance in general and to a Tissue Sampling and RNA Extraction later crossing point. Therefore, the extracted total RNA quality and quantity must be verified prior to qRT-PCR experiments to The sampling and preparation of intact cellular total RNA or end in reliable mRNA quantification results. mRNA is critical to all gene expression analysis techniques. A successful and reliable experiment needs high quality, DNA free, undegraded RNA. The source of tissue sampling techniques and the subsequent storage of the tissue material often varies significantly between processing laboratories (e.g. biopsy material, single cell sampling, laser micro-dissection, slaughtering samples). Some researchers store the tissue sample in RNAlater®

Our Innovation, Your Research — Shaping the Future of Life Science 35 36

Appendix 2 variability during multiple RT reactions.variability duringmultipleRT have thesamecDNAsynthesisefficiencies.The result canbehigh gene ofinterest. Itcannot be assumedthatdifferent reactions RT gene-specific primersnecessitatesaseparate reaction foreach gene-specific senseprimer(forward primer);however, theuseof subsequent PCRassayinconjunctionwiththecorresponding spurious transcripts.Thesamereverse primerisusedforthe reactionconjunction withelevatedRT temperatures toeliminate specific ornon-specific. Targetgenespecificprimersworkwell in used toinitiatecDNAsynthesis,whichcanbeeithertargetgene- Another source ofvariabilityisthechoiceprimingmethod specificity andefficiencyoffirstprimerannealing. minus RT maintains its activity up to 70 °C, thus permittingthe AMV increasedonly around 5-20%. Newly available to reversethermo-stable transcribebetween50and80%ofthetotalRNA RNAse H than thatofAMV. Innumbers,theMMLV hastheability HminusRT of choice,asthecDNAsynthesisratecanbeupto10-foldgreater For many quantitative applications, MMLV H minus RT is the enzymereaction efficienciesandgenerateunreliable quantification results. enzymatic tissueinhibitorsthatresult inreduced andPCR RT efficiency.RT TheextractedtotalRNAmaycontainchemicalor carried overfrom theRNAisolationprocess canaffect theapparent fatty acids,alcohol,phenolandotherchemicalorenzymeinhibitors conditions havetobeoptimised.Buffer saltcontamination,pH, in akineticRT-PCR experimentandforeachenzymespecific reaction, successful qRT-PCR assay. stepisthesource TheRT ofhighvariability An optimalreverse isessentialforareliable transcription(RT) and Reverse Transcription (41 s)andadominant28SRNApeak(43s). reference peak(22s),asmall5SRNA(27dominant18S ratio 28s/18s=1.4).ThecharacteristictotalRNAprofile represent aninternal An electropherogram oftotalRNAisshown(178ng/µl totalRNA;RIN=7.9;: Appendix 2 Figure 2 sigma.com ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

in the4 elevated temperatures (herein 85°C).Thefluorescence acquisition specific primer(M.Pfaffl,unpublished results). specific gene:randomhexamerprimers>poly-dTprimergene efficiency canbeshownfortheapplieddifferent primersforone pool.Inconclusion,arankorderone andthesameRT ofRT directly comparablebetweentheappliedassays,atleastwithin one smallRNAsample.Therefore, thegeneexpression results are genes thatcanbeassayedfrom asinglecDNApoolderivedfrom target-specific kineticPCRassays.Thismaximisesthenumberof with unspecificprimerscanbesplitintoanumberofdifferent 3-’ (poly-A)tailfoundonmostmRNAs.cDNApoolssynthesised desoxythymidine residues) canannealtothepoly-adenylated Similarly, poly-Toligo-nucleotides(consistingsolelyof16-25 primers, canbeusedandacDNApoolsynthesised. unspecific primers,e.g.random-hexamer, -octameror-decamer To circumvent thesehighinter-assay variationsinRT, targetgene an additional4 structures, atleastduringthequantificationprocedure, istoadd The easiestandmosteffective waytogetridofanydimer optimisation strategieshavebeendeveloped. primer pairbycross-wise combinations.Multiplesofsuchprimer an intensiveprimeroptimisationisneededbytestingmultiple and havediffuse andbroader peaks.To getridofprimerdimers, contrast, theprimerdimersmeltatlowertemperatures (<80°C) a single,sharplydefinedmeltingcurvewithnarrow peak.In software. Thepure andhomogeneousRT-PCR product produces curve analysiscanbeperformedinallavailablequantification distinguish primerdimersfrom thespecificamplicons,amelting RTPCR efficiency andalesssuccessfulspecific product. To of specificPCRproduct, leadingto reduced amplification During PCR,formationofprimerdimerscompeteswith take placeassoonPCRreagents are combinedin the tube. specific annealingandprimerelongationevents.Theseevents the presence ofprimerdimers,whichare theproduct ofnon- Real-time assaysusingSYBRGreen Ibindingdyecaneasilyreveal Elevated Fluorescence Acquisition elongation at72°C;4 procedure, Figure 3:1 2 cence range. fluorescence atplateauand ensures anoptimaldynamicfluores- and the ‘no-template control’ fluorescence under 10% of maximal temperature quantification keepsthebackground fluorescence accurate quantificationofthedesired qRT-PCR product. High derived byprimerdimersorunspecificminorproducts andensures nd segmentwithprimerannealingat60°C;3 th segmenteliminatesthenon-specificfluorescence signals th segmenttotheclassicalthree segmentedPCR st th segmentwithdenaturationat95°C; segmentwithfluorescence acquisitionat rd segmentwith

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Figure 3 acceleration of fluorescence. The amplification reaction and the kinetic fluorescence history over various cycles is obviously not a 100 72 °C smooth and easy function. The mathematical algorithm on which 50

the “second derivative maximum method” is unpublished, but it Appendix 2 20 log) 10 is possible to fit sigmoidal and polynomial curve models, with 3-step vs. 4-step 10 high significance, p<0.001, and coefficient of correlation, r>0.99. real-time qPCR negative control fluorescence ( 5 10^9 molecules 10^8 molecules This increase in the rate of fluorescence increase, or better called 2 10^7 molecules 10^6 molecules 10^5 molecules the acceleration of the fluorescence signal, slows down at the 1 10^4 molecules 0510215205 30 rd cycles 10^3 molecules beginning of the 3 linear phase. Therefore the cycle where the 10^2 molecules nd nd 100 85 °C 10^1 molecules 2 derivative is at its maximum is always between 2 exponential rd 50 and 3 linear phase. Here, above the background level within the 85 °C 20 real exponential phase and very close to the background line, the log) 10

10 optimal CP should be placed.

72 °C fluorescence ( 5

2

1 051015220530354045 50 cycles

Advantage of elevated real-time PCR fluorescence acquisition temperature.

Crossing Point Data Evaluation The amount of amplified target is directly proportional to the input amount of target only during the exponential phase of PCR amplification. Hence the key factor in the quantitative ability of kinetic RT-PCR is that it measures the product of the target gene within that phase. The data evaluation, or crossing points (CP) or threshold cycle (Ct) determination is very critical to the users. For CP determination, various fluorescence acquisition methodologies are possible. The “fit point method” (e.g. in LightCycler software) and the “threshold cycle method” (used in most quantification software) measure the CP at a constant fluorescence level. These constant threshold methods assume that all samples have the identical synthesised DNA concentration at the point where the fluorescence signal significantly increases over the background fluorescence, 2nd to 3rd phase in Figure 1. Measuring the level of background fluorescence can be a challenge in real-time PCR reactions with significant background fluorescence variations, caused by drift-ups and drift-downs over the course of the reaction. Averaging over a drifting background will give an overestimation of variance and thus increase the threshold level. The threshold level can be calculated by fitting the intersecting line upon the ten-times value of ground fluorescence standard deviation, e.g., Applied Biosystems software. This acquisition mode can be easily automated and is very robust. In the “fit point method” the user has to discard the uninformative background points, exclude the plateau values by entering the number of log-linear points, and then fit a log-line to the linear portion of the amplification curves. These log lines are extrapolated back to a common threshold line and the intersection of the two lines provides the CP value. The strength of this method is that it is extremely robust. The weakness is that it is not easily automated and so requires a lot of user input. The problems of defining a constant background for all samples within one run, sample-to- sample differences in variance, and absolute fluorescence values led to the development of new acquisition modus according to mathematical algorithms. In the LightCycler software the “second derivative maximum method” is performed in which CP is automatically identified and measured at the maximum

Our Innovation, Your Research — Shaping the Future of Life Science 37 38

References 15.  14.  13.  12.  11.  10.  9. 8. 7. 6. 5. 4. 3. 2. 1. References 28, 732-738(2000). the 5’-nucleaseTaqMan assayandMolecularBeaconprobes. Biotechniques, Tapp, I.,etal.,Scoringofsingle-nucleotidepolymorphisms:comparison Molecular Beacons.Genet.Anal.,14,151-156(1999). Marras, S.A.,etal.,Multiplexdetectionofsingle-nucleotidevariationsusing automated mutationanalysis.Clin.Chem.,44,482-486(1998). Giesendorf, B.A.,etal.,MolecularBeacons:anewapproach forsemi­ nucleotide polymorphismswithreal-time PCR.Methods,25,463-471(2001). Mhlanga, M.M. and Malmberg, L., Using Molecular Beacons to detect single- Beacons. Anal.Biochem.,300,40-45(2002). Liu, J.,etal.,Real-timemonitoringinvitrotranscriptionusingMolecular pathways inlivingcells.Histochem.CellBiol.,115,3-11(2001). Dirks, R.W., etal.,MethodsforvisualizingRNAprocessing andtransport ization insidesinglelivingcells.Anal.Chem.,73,5544-5550(2001). Perlette, J.andTan, W., Real-timemonitoringofintracellularmRNAhybrid- tion andemergingapplications.J.Biomol.Struct.Dyn.,19,497-504(2001). Antony, T. andSubramaniam,V., MolecularBeacons:nucleicacidhybridiza - hybridization. Nat.Biotechnol.,14,303-308(1996). Tyagi, S.andKramer, F.R., MolecularBeacons:probes thatfluoresce upon ogy andbiotechnology. Trends Biotechnol.,20,249-256(2002). Broude, N.E.,Stem-loopoligonucleotides:Arobust tool formolecularbiol- structured DNAprobes. Proc.Natl.Acad.Sci.USA,96, 6171-6176 (1999). Bonnet, G.,etal.,Thermodynamicbasisoftheenhancedspecificity the LightCycler. J.Biochem.Biophys.Methods,47,121-129(2001). Pals, G.,etal.,Detectionofasinglebasesubstitutionincellusing 300-334 (1995). Selvin, P.R., Fluorescence resonance energytransfer. Meth.Enzymol.,246, BRCA1 asanexample.MolDiagn.,4,241-246(1999). real-time polymerasechainreaction andmeltingcurve analyses,using Pals, G.,etal.,Arapidandsensitiveapproach tomutationdetectionusing fication reactions. Biotechnology, 11,1026(1993). Higuchi, R.,etal.,KineticPCRanalysis:Real-timemonitoringofDNAampli- sigma.com ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832 27.  26.  25.  24.  23.  22.  21.  20.  19.  18.  17.  16.  (2002). PCR (RT-PCR): trends andproblems. J.MolecularEndocrinology, 29,23-29 Bustin, S.A.,QuantificationofmRNAusing real-time reverse transcription recognition ofDNAandRNA. Chem.Biol.,8,1-7(2001). Braasch, D.andCorey, D.,Lockednucleicacid(LNA):fine-tuning the nucleic acidprimers.Biotechniques,34,150-152,1154,1158(2003). Latorra, D.,etal.,Multiplexallele-specificPCRwithoptimizedlocked tions. Biochem.J.,354,481-484,(2001). nucleotide duplexformation:studiesofLNA-DNAandDNA-DNAinterac- Christensen, U.,etal.,Stopped-flowkineticsofLockedNucleicAcidoligo- Molecular andCellularProbes,17,253-259(2003). Latorra, D.,etal.,Designconsiderationsandeffects ofLNAinPCRprimers. modified RNA.Biochem.,42,7967-7975(2003). Braasch, D.,etal.,RNAinterference inmammaliancellsbychemically- 3204-3212 (2001). of humanpapillomavirusesinclinicalsamples.J.Clin.Microbiol.,39, Hart, K.W., etal.,Novelmethodfordetection,typing,andquantification mutation detection.NucleicAcidsRes.,28,3752-3761(2000). Thelwell, N.,etal.,ModeofactionandapplicationScorpionprimersto cons andfluorescence. Nat.Biotechnol.,17,804-807(1999). Whitcombe, D.,etal.,DetectionofPCRproducts usingself-probing ampli - tions. NucleicAcidsRes.,29,E96(2001). Solinas, A.,etal.,DuplexScorpionprimersinSNPanalysisandFRETapplica- Molecular Beacons.Proc.Natl.Acad.Sci.USA,96,6394-6399(1999). Vet, J.A.,etal.,Multiplexdetectionoffourpathogenicretroviruses using Reprod., 5,1034-1039(1999). embryos byreal-time rapidcyclefluorescence monitoredRT-PCR. Mol.Hum. Steuerwald, N.,etal.,Analysisofgeneexpression insingleoocytesand INNOVATION @ WORK

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