technology Manipulating a troubleshooting guide: EXPERTS share their advice ON MEASURING microRNAs

methods

Table of Contents

Letter from the Editor ...... 5 Index of Experts ...... 5 Q1:  How do you effectively isolate and purify miRNA from cells?...... 6 Q2:  How do you efficiently clone an miRNA? ...... 7 Q3: What method do you use to ensure sensitive and specific gene expression profiling of miRNAs? Why?. . . . 8 Q4: How do you validate your expression results so that you get robust and reproducible data? ...... 10 Q5:  What method do you use to up- or down-regulate a specific miRNA? Why? ...... 11 Q6:  How do you verify the results of up- or down- regulating an miRNA? ...... 12 List of Resources ...... 14

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Letter from the editor This month, GT brings And while the toolsets for working with you a technical guide on miRNAs — overexpressing them or knock- manipulating microRNAs. ing them out to see what ensues — have Although the first miRNA greatly improved during the past few years, was discovered as recently there are still tricks to master. as 1993, scientists have To that end, we’ve consulted some of the learned much about how best and brightest in the field to get feed- these small, noncoding affect gene ex- back on how best to perform both dis- pression. Normally, they down-regulate genes covery and expression profiling studies. by interacting with expressed transcripts, ef- Their troubleshooting advice ranges from fectively turning them off or dampening their how to isolate and purify miRNA from expression. Recent work has shown them to cells, to how to up- or down-regulate a influence at least 30 percent of genes. specific miRNA and how to verify the re- Not only have miRNAs been found to play sults. We hope this steers you in the right an important role in regulating development direction. And for even more thought- as well as protecting against cancer, they are ful instruction, check out our resources differentially expressed in tissues, making guide at the back. expression profiling a key area of research. — Jeanene Swanson

Index of experts Winston Kuo Brigham and Women’s Hospital, Many thanks to our experts for taking the time Harvard UniverSIty to contribute to this technical guide, which would not be possible without them.

Galina Gabriely Joshua Mendell Brigham and Women’s Hospital, Johns Hopkins University Harvard UniverSIty

Peng Jin Silvia Monticelli Emory University Institute for Research in Biomedicine, Switzerland

Manipulating MicroRNAs SEPTEMBER 2009 genome technology 5

How do you effectively isolate and purify miRNA from cells?

For miRNA isolation we routinely it requires the addition of a lysis ply gel-purify small RNAs from use TRIzol RNA reagent, which is reagent .A critical issue with RNA TRIzol-isolated total RNA . normally used for total RNA iso- sample preparation is the effec- — Joshua Mendell lation . This method is sufficient tive precipitation of small RNA If you are not limited with the for purification of miRNAs from fractions . Traditional meth- amount of starting material (i e. ., cultured cell lines . However, if ods using 70 percent ethanol cells or tissues), then a regular isolation is required from small washes do not effectively pre- TRIzol extraction of total RNA is or poor-quality tissue samples, cipitate small RNAs . Increas- enough, and you get good qual- we use the mirVana miRNA Iso- ing the ethanol concentration ity and reproducible yields . We lation Kit from Ambion .With this to >80 percent in these steps routinely use RNA purified in kit, miRNA could be isolated us- will address this problem . this way to detect miRNAs by ing either total RNA extraction — Winston Kuo northern blot and real-time PCR, protocol or enriched for small For expression analysis in cells with very comparable and repro- RNAs/miRNA . ducible results . It is a bit trickier and tissues, straightforward to- — Galina Gabriely when you have smaller amounts tal RNA isolation methods work of starting material . You can still In general we use TRIzol to iso- well . We typically use TRIzol . just use TRIzol, but you need to late RNAs from cells . TRIzol has More sophisticated column- improve the RNA precipitation been very effective to recover based methods or further purifi- step, and we found that adding small RNAs from tissues or cells . cation of small RNA populations some glycogen-blue works just — Peng Jin has not been necessary in our fine . Otherwise you can enrich experience . Furthermore, DNA for the small-RNA fraction using In general, several techniques contamination, which can some- spin-column kits like the mirVana have been tried in our lab, and times be an issue when assess- from Ambion . this depends on the method we ing mRNA abundance in TRIzol- — Silvia Monticelli would use for profiling . TRIzol isolated RNA, does not seem to has been very effective and the be a problem when measuring mirVana miRNA Isolation kit miRNAs in these samples . “For miRNA from Ambion . Other methods If we wish to specifically isolation we use can also be used effectively . For analyze small RNA populations TRIzol reagent.” certain technologies, there is no in cells (e g. ,. for sequencing need for isolation or purification; small RNA libraries), we sim- — Galina Gabriely

6 TEch guide SEPTEMBER 2009 Manipulating MicroRNAs

How do you efficiently clone an miRNA?

It depends on the purpose cloning of a much smaller se- RNA polymerase II promoter) of cloning an miRNA . If it is quence encoding the miRNA and robust expression will be for miRNA discovery or high- only with little or no flanking achieved with rare exception . throughput sequencing, we will sequence, this approach to to directly clone mature use 5’- and 3’-adapter ligation miRNA cloning is highly ef- miRNA species (e .g ., to identi- to generate small RNA librar- ficient . The same protocols fy novel miRNAs), small RNAs ies . If it is a known miRNA we that are used to generate are first isolated from total are interested in expressing, shRNA hairpin vectors work RNA by gel purification . After we generally use PCR to ampli- well for miRNA cloning . You ligating on adapters, small fy the DNA fragment contain- simply have oligos encoding RNA populations are ampli- ing the miRNA of interest and the miRNA synthesized that fied by PCR and then directly then verify its expression from contain the appropriate over- sequenced using next-gener- ation sequencing technology an appropriate vector . hangs for cloning and per- (i e. ., 454, Solexa, SOLiD) . — Peng Jin form an annealing reaction — Joshua Mendell followed by a ligation reaction When cloning an miRNA, there into the linearized vector . This Our lab doesn’t really have are a couple of issues . If you strategy is often used when much direct experience in this . are interested in cloning a large straight overexpression is re- But there are many protocols chunk of the genomic context quired for the experiment and out there that are widely avail- surrounding the miRNA (e .g ., endogenous / able and very accurate, and upstream promoter/enhancer re- processing is not relevant or from labs that have done excel- gions), this method is often used even undesirable . lent work in the field .The Bartel when endogenous transcrip- — Winston Kuo lab, for example, has posted on tional regulation and processing its website very detailed proto- (e .g ., Drosha/Dicer action) is un- To clone an miRNA-encoding cols to work on miRNAs . der examination . In this instance, sequence from genomic DNA, — Silvia Monticelli standard PCR-based methods we simply PCR-amplify the pre- can be used to generate miRNA miRNA hairpin and ~100 bp of inserts with compatible restric- 5’ and 3’ flanking sequence . “It depends on tion enzyme sites for your vector These fragments can be directly the purpose.” system of interest . cloned into virtually any mam- If you are interested in malian expression vector (with — Peng Jin

Manipulating MicroRNAs SEPTEMBER 2009 genome technology 7

What method do you use to ensure sensitive and specific gene expression profiling of miRNAs? Why?

We usually perform small-scale microRNA array platform that sensitive manner . profiling of small numbers of contains probes with locked nu- — Winston Kuo human miRNAs using multi- cleic acid-enhanced oligonucle- plex real-time PCR .This method otide capture probes . LNA are In my laboratory, we are cur- provides better sensitivity and modified in which rently using custom-spotted specificity than microarray the ribose ring is “locked” with microarrays profiling and therefore is pref- a methylene bridge connect- primarily because they are erable when the number of ing the 2’-O atom and the 4’-C cost-effective and easy to use . miRNAs in question is not very atom . This increases the melt- For greater sensitivity, there are high . The accuracy of profiling ing temperature of the duplex good commercially-available by multiplexed RT-PCR can be 2-8 degrees C per LNA mono- options such as the Exiqon and validated by more sensitive, mer . This vastly improves the Agilent microarrays and the singleplex reaction . thermal stability and specificity Applied Biosystems TaqMan — Galina Gabriely of duplexes formed with com- arrays (which use real-time plimentary miRNA sequences . We generally utilize an miRNA PCR for profiling) . Incorporation of LNA nucleo- TaqMan assay to determine the — Joshua Mendell sides also allows for the gener- expression of specific miRNAs . ation of Tm-normalized capture We have tried multiple ap- After years of northern blots and probes . This is very important proaches and found that both dot blots to profile microRNA for the performance of the mi- sensitivity and specificity of expression, more and more croarray, as miRNA are short TaqMan are better . Of course, frequently we use the miRNA and have a broad Tm range . we also use high-throughput TaqMan assays from Applied Luminex’s FlexmiR microRNA sequencing for digital gene Biosystems, that are extremely panels combine Exiqon’s LNA expression profiling of small specific and sensitive, and the probes to achieve high speci- RNAs, which has good correla- data always correlate very well ficity . HTG, on the other hand, tion with TaqMan assays . with our northern blots . uses complementary nucle- — Peng Jin — Silvia Monticelli ase protection probes that We have used multiple miRNA are hybridized to the miRNA profiling technologies that in- in the sample, and then an S1 “We use TaqMan clude those from Exiqon, Febit, endo/exo nuclease reaction assays.” High Throughput Genomics, In- is carried out which destroys vitrogen, and Luminex .Exiqon’s mismatched probes in a very — Silvia Monticelli

8 TEch guide SEPTEMBER 2009 Manipulating MicroRNAs

How do you validate your expression results so that you get robust and reproducible data?

We use qRT-PCR with TaqMan cumvents the issues presented we can use the delta delta Ct rela- microRNA assays and normal- by the observed endogenous tive quantitation method . That is ize the data on other miRNAs 3’-end miRNA sequence diver- the assays for our miRNA(s) of which are known to be stable . sity . This diversity is a problem interest and the normalization One can also use snoRNAs for for the TaqMan stem-loop RT- control(s) need to have similar normalization . In any case, we primer strategy . From a techni- (<10 percent difference) slopes usually normalize on two to cal standpoint a single oligo dT as obtained from the 5X dilu- three different molecules to en- RT reaction (Qiagen) is superior tion series . We start with five sure the robust estimation of (cost, liquid handling, variability) controls (U6B snoRNA, snoR- miRNA amounts . Also, to ensure to the unique stem-loop RT re- NA24, snoRNA49, GAPDH, robust and reproducible data, actions (TaqMan) that must be and select the one to two most we perform experiments with performed for each individual appropriate assays) . biological repeats . miRNA of interest . We perform 2 . Northern blotting: We use — Galina Gabriely qPCR assays in 384-well plates radiolabelled LNA-based oligos as with four technical replicates/ probes for miRNA northern blots . By comparing different assay sample and three experimental Northerns are run in triplicate formats early on, we have found replicates per treatment group (three experimental samples per that miRNA TaqMan assay is very (12 total wells per condition) to treatment group) to demonstrate robust and reproducible . For the get robust and reproducible data . reproducibility . Many miRNAs miRNAs with high abundance, Appropriate no-template and no are found in functional-related we also confirm the expression primer controls are included . families (share 100 percent seed data by traditional northern blot . We perform all the normal sequence homology and variable — Peng Jin steps to optimize any qPCR assay . 3’-end homology) . Therefore, Pilot experiments are performed when using qPCR or northerns We validate our expression to demonstrate the assays are you must prove that under your results using three compli- linear (5X dilution series) and to conditions the assay only detects mentary strategies: ensure that they yield a single your miRNA of interest and not 1 . qPCR: There are two ap- product (melting curve analysis) . other family members . We do proaches we have used, the We also perform an extensive this by spiking synthetic mature miScript SYBR Green System normalization control selection miRNAs (IDT) into a corn (Zea) RNA (Qiagen) and TaqMan miRNA experiment to ensure that 1) the carrier background . assays (ABI) . We prefer Qiagen’s normalization control does not for PCR each synthetic strategy because it involves a change in our treatment groups miRNA family member (in- poly-A tailing approach that cir- and/or cell lines of interest and 2) put) is run against a panel of continued on page 13

10 TEch guide SEPTEMBER 2009 Manipulating MicroRNAs

What method do you use to up- or down-regulate a specific miRNA? Why?

For down-regulation of miRNA expression vectors containing a man cancer cell lines (the model we use antisense oligonucle- pri-miRNA DNA fragment to systems we work in most often) . otides (ASO) modified with overexpress a specific miRNA . Furthermore, Exportin 5 has 2’-O-metoxyethylribose (MOE) To down-regulate a specific been shown to be a bottleneck in provided by our collaborators miRNA, we will either express the miRNA biogenesis pathway (Regulus), as they result in the a shRNA that could produce in cell and mouse models . Exog- most specific inhibition . In addi- siRNA against the stem-loop enous overexpression involving tion, we use commercially avail- region of an miRNA precursor DNA integration can saturate Ex- able locked (LNA)- or use a miRNA sponge that portin 5 thus competing off en- modified oligos . Due to their could express a reporter gene dogenous small RNA sequences . high-affinity binding to miRNAs, with multiple target sites of In extreme cases this can lead LNA-modified oligos provide specific miRNA . to cell/animal death; however, strong inhibition, though they — Peng Jin we presume that less penetrant may have some off-target ef- phenotypes are also possible fects . For up-regulation we use There are two general strategies: and could result in serious exper- synthetic miRNA mimics avail- 1) vector/viral-based overexpres- imental artifacts . Transfection of able through Ambion . sion of miRNAs or miRNA anti- exogenous duplexes that enter — Galina Gabriely sense sequences and 2) trans- the pathway via direct incorpo- fection of exogenous miRNA ration into the RISC complex cir- We use both RNA oligo- and duplexes or antisense inhibitors . cumnavigate these pitfalls . vector-based approaches . For The method chosen depends on Of course, in cases where an RNA oligo-based approach, the design of the experiment . persistent gain-of-function we apply siRNA duplex-like generally speaking, we fa- or loss-of-function is required miRNA and antimir/antagomir to vor transfection of exogenous (e .g ., xenograft models of tu- increase or block the activity of gain-of-function and loss-of- mor growth) stable expres- a specific miRNA .This approach function reagents . Vector/viral- sion using vector/viral-based is very convenient for manipulat- based strategies often rely on solutions are required . Stable ing the activity of specific miRNA sequential processing of the lines must be established in cell culture . resulting transcripts by Drosha/ carefully, employing viral ti- A vector-based approach Dicer and must be exported tering experiments to avoid has the advantages of long-term from the nucleus via Exportin 5 . the pitfalls descibed above . alteration and the ability to track miRNA processing defects at the — Winston Kuo individual cells . We utilize arti- level of Drosha and Dicer have ficial shRNA or Pol II been reported in established hu- For upregulating miRNA, we continued on page 13

Manipulating MicroRNAs SEPTEMBER 2009 genome technology 11

How do you verify the results of up- or down-regulating an miRNA?

To assess miRNA activity fol- allow for direct detection of to monitor miRNA inhibition lowing its manipulation, we miRNA overexpression or down- is to use a reporter construct measure expression of lu- regulation . Luciferase reporter with an miRNA binding site . The ciferase reporter fused to the assays involve cloning of 3’-UTR miRNA will inhibit expression of perfect binding site of the sequences downstream of the the reporter unless its activity miRNA . However, the best luciferase coding sequence . is blocked . Of course, the use of way to evaluate the activity These 3’-UTRs can contain ar- such reporters is generally lim- of miRNA is to assess the ex- tificial miRNA cognate sites or ited to a cell culture setting . pression of its natural targets endogenous sequences from — Joshua Mendell (when the target of an miRNA validated mRNA targets (when is known) . This should be this information is available) . A This is a critical step in these done by western blot analysis . critical control in the later strat- kinds of experiments: by trans- Modulation of miRNA expres- egy is the generation of point fecting or transducing cells sion can be assessed by mea- mutants in the seed sequence you are effectively somehow suring the expression levels of putative cognate sites . Such changing them, so you really of miRNA using northern blot mutants should abrogate the need a lot of controls to make analysis . In some cases, qRT- ability of the miRNA to regulate sure that the effect you are PCR with TaqMan microRNA luciferase expression . seeing is really due to miRNA assays may also give reliable — Winston Kuo expression or down-regulation . results for miRNA expression, It is particularly important but oligos used for miRNA To verify miRNA up-regulation, with miRNAs because in many modulation may interfere with we again use northern blotting cases you don’t really expect primers during PCR reaction, or real-time PCR to measure a strong phenotype, but you which often affects the real miRNA abundance . Measuring are looking for milder effects of expression data . miRNA inhibition may not be as ‘fine-tuning’ gene expression . — Galina Gabriely simple . Often, antisense oligo- I think you always need to use do reduce the abun- at the very least scrambled oli- We use several strategies in- dance of the miRNAs they tar- gos, miRNAs different from the cluding miRNA qPCR, miRNA get . So one can use these same one you are interested in and northern blot, luciferase reporter methods to monitor miRNA in- in the seed region . I assays, mRNA target qPCR, hibition . However, sequestering would also recommend using and target western blot . an miRNA in vivo may not always different approaches to try to I discussed miRNA qPCR and result in a decrease in miRNA achieve the same results . Northern blotting above . These abundance . An alternative way — Silvia Monticelli

12 TEch guide SEPTEMBER 2009 Manipulating MicroRNAs Q4: continued from page 10 once the appropriate qPCR precious) . We have found that qPCR assays for each miRNA and/or northern blotting condi- both methods produce high- family member (assay) . The tions are established using the quality expression data when value for the correctly paired spike-in strategy they can be ap- performed properly . Northern input x assay combination is plied to experimental samples . blotting is cheaper but requires set to 100 percent relative 3 . In situ hybridization: We a lot of RNA, whereas real-time detection and mismatched also have used Exiqon’s DIG- PCR is more expensive but input x assay combinations labelled miRCURY LNA microRNA very sensitive . are expressed relative to detection probes to confirm our — Joshua Mendell 100 percent . Thermocycling miRNA expression results and conditions can be modified tissue specificity . We developed I am old-fashioned, I like to see as needed to reduce assay a working in situ hybridization bands on a gel! We make sure cross-talk with other miRNA protocol on our frozen mouse that expression data are always family members . 18 5. d p. c. . embryonic cranial consistent using different kinds In the case of northerns, coronal sections . of approaches . Obviously this is synthetic miRNAs (in the car- — Winston Kuo not always possible, as, for ex- rier RNA) for each of the miRNA ample, for northern blot you need family members is run on a 15 Careful validation of all microar- 25-30 mg of total RNA per lane percent PAGE gel containing ray results are very important, (versus 10 ng for the TaqMan- 8M urea . Hybridization tem- regardless of the platform used . based approaches) and you just peratures are optimized so that We generally use either north- don’t always get this much RNA, the LNA probe only binds to the ern blotting (when we have lots especially if you are working with miRNA of interest and not the of RNA to work with) or real- primary cells . other family members . time PCR (when the RNA is very — Silvia Monticelli

Q5: continued from page 11 ologic expression levels are often miRNA binding sites that are often construct expression achieved, so care must be taken thought to sequester the active vectors (in standard mamma- in interpreting results from these miRNAs in a cell . lian expression plasmids or viral experiments . — Joshua Mendell vectors) . Making these is very to inhibit miRNAs, we gen- simple . As described above, erally use commercially avail- We use mostly lenti- or retrovirus- we simply amplify the miRNA able antisense . based transduction systems hairpin and some flanking ge- We have had success with the because we work mainly with nomic sequence and clone the inhibitors made by Dharmacon primary cells and we need sus- PCR products directly . Alterna- and Exiqon . The limitation of tained expression for long periods tively, we sometimes transiently these reagents is that they only of time . Depending on the cell type, transfect cells with synthetic work transiently . To achieve we also use transient transfec- miRNA mimics (RNA oligonu- stable inhibition, we have tion with lipofectamine or Amaxa . cleotides identical in sequence started working with so-called For down-regulation we are using to the mature miRNA) to over- “miRNA sponge” constructs, as miRNA sponges . For short-term express an miRNA . With both described first by Phil Sharp’s experiments transient transfection of of these methods ( vs . laboratory . These are essentially antagomirs works well, too . synthetic mimic), supraphysi- decoy transcripts with multiple — Silvia Monticelli

Manipulating MicroRNAs SEPTEMBER 2009 genome technology 13 List of resources Our panel of experts referred to a number of tools that may lin-14 . Cell . 1993 Dec 3;75(5):843-54 . be able to help you get a handle on working with miRNAs. Whether you’re a novice or pro at manipulating microRNAs, Lim LP, Lau NC, Garrett-Engele P, Grimson A, these resources are sure to come in handy. Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson Jm . Microarray analysis shows publications that some microRNAs downregulate large Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, numbers of target mRNAs . Nature . 2005 Feb 17;433(7027):769-73 . Epub 2005 Jan 30 . West KM, Dang CV, Thomas-Tikhonenko A, Mendell Jt . Widespread microRNA repression Marson A, Levine SS, Cole MF, Frampton GM, by Myc contributes to tumorigenesis . Nat Brambrink T, Johnstone S, Guenther MG, John- Genet . 2008 Jan;40(1):43-50 . Epub 2007 Dec 9 . ston WK, Wernig M, Newman J, Calabrese JM, Dennis LM, Volkert TL, Gupta S, Love J, Hannett N, Johnson SM, Lin SY, Slack FJ . The time of ap- Sharp PA, Bartel DP, Jaenisch R, Young RA . Con- pearance of the C. elegans let-7 microRNA necting microRNA genes to the core tran- is transcriptionally controlled utilizing a scriptional regulatory circuitry of embryonic temporal regulatory element in its promoter . stem cells . Cell . 2008 Aug 8;134(3):521-33 . Dev Biol . 2003 Jul 15;259(2):364-79 . Monticelli S, Ansel KM, Xiao C, Socci ND, Krichevsky Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, AM, Thai TH, Rajewsky N, Marks DS, Sander C, Tuschl T, Manoharan M, Stoffel m . Silencing of Rajewsky K, Rao A, Kosik ks . MicroRNA profiling microRNAs in vivo with ‘antagomirs’ . Nature . of the murine hematopoietic system . Genome 2005 Dec 1;438(7068):685-9 . Epub 2005 Oct 30 . Biol . 2005;6(8):R71 . Epub 2005 Aug 1 . Lagos-Quintana M, Rauhut R, Yalcin A, Meyer Vella MC, Choi EY, Lin SY, Reinert K, Slack FJ . The J, Lendeckel W, Tuschl t . Identification of C. elegans microRNA let-7 binds to imperfect tissue-specific microRNAs from mouse . let-7 complementary sites from the lin-41 Curr Biol . 2002 Apr 30;12(9):735-9 . 3’UTR . Genes Dev . 2004 Jan 15;18(2):132-7 . Epub Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino 2004 Jan 16 . N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, websites Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, miRBase Müller RU, Schermer B, Bissels U, Inman J, Phan http://microrna sanger. .ac .uk/sequences Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann MicroRNA.org G, Palkovits M, Di Lauro R, Wernet P, Macino http://www .microrna org/microrna/home. do. G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Target Gene Prediction at EMBL Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio http://www .russell embl. de/m. iRNAs A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl t . A mammalian microRNA expression atlas based on small RNA library sequencing . conferences Cell . 2007 Jun 29;129(7):1401-14 . MicroRNA and Cancer Keystone Symposia on Molecular and Cellular Biology Lee RC, Feinbaum RL, Ambros v . The C. elegans heterochronic gene lin-4 encodes small MicroRNA in Human Disease & Development RNAs with antisense complementarity to Cambridge Healthtech Institute

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