Europe -Diagenodes.a. PROTOCOL homogenization: Bioruptor disrupting while RNA of cells anddissolvingintegrity cell components. the maintains RNA and the medium sonication in as (included used reagentis kit) extraction extraction RNA Diagenode’s purification. step. one RNA in tissues facilitate homogenize and to viscosity sample reduces Diagenode’sBioruptor homogenization while RNA, releases disruption and homogenization of animal tissues are required to ensure high yield of expressionEfficient analysis.RNA. gene in Disruption used techniques many for essential is RNA intact of Isolation Introduction using Bioruptor RNA extraction from tissue - General remarks before starting • • • • • • • • • • /orders s t ih rsl i lw ult RA Fr agr uniy ct h tsu ad rce to proceed and tissue the cut quantity, larger For RNA. quality low in result might it as samplemoretissueper use not tissue.Do of mg protocolvalidatedfor20-50 This been has RNA until -80°C at stored and nitrogen liquid extraction. Alternatively, RNAlater solutioncan beusedto protect RNAinunfrozen sample. in snap-frozen be can tissues Dissected to correlates quality tissue-specific response to physiological stress RNA bothprior to and following tissue from death. manipulation. and sample by RNases triggered expression by RNA degradation in RNA changes prevent to collection tissue of time the Minimize Multiplexing capability ofupto 6samples inparallel (dependsonBioruptor Isothermal process Efficient and reproducible Non-contact minimizescontamination Fast protocol ice whenaliquotsare pipetted for downstream applications. equipment. laboratory dusty on RNA fromisolated Keep possible.whenever closed or tubes keep frequentlyand gloves Change skin the of surface the from contamination RNase prevent to times all at gloves Wear analysis. through continuing and purification RNA with When working with RNA, care must be taken to maintain an eye RNase-free environment and starting gloves use and hood fume protection. in work reagent, extraction RNA with working When disruption inseparate tubes. ® @ together with RNA extraction reagent offer unique benefits for tissue disruption and disruption tissue for benefits unique offer reagent extraction RNA with together diagenode.com /info ® Sonicator uses state-of-the-art ultrasound technology to efficiently disrupt efficiently technologyto state-of-the-artultrasound uses Sonicator @ diagenode.com // ® (Standard/Plus) andRNAextraction kit North America -Diagenode Inc. /orders.na @ diagenode.com /info.na ® model) @ diagenode.com www.diagenode.com 1 Europe -Diagenodes.a. PROTOCOL 1. Tissue disruptionandhomogenization Protocol Required materials andreagents 8. 7. 6. 5. 4. 3. 2. • • • • • • • • • • • • /orders If needed, cut the frozen tissue in a Petri dish placed on dry ice. Minimize the time required to do this, and and this, do to required time the Minimize ice. dry on placed dish Petri a in tissue frozen the cut needed, If RNAlater-treated samples can beused. Alternatively,reagent. extraction RNA containing tube per tissuesnap-frozen of mg 20-50 Add tubes Keep thetubesonice. these use to recommend highly We tubes. Prepare sonication tubes: add 1 ml of cold RNA extraction reagent to the pre-filled RNA extraction ice. BioruptorPre-coolthe using 4°C toBioruptor sample for disruption. Bioruptor Stop Sonicate samples inBioruptor using thefollowing settings: during holders shouldalways becompletely holder filled withtubes. tube in tube the tube sonication, of the homogeneity of guaranteeTo picture). position (see sonication optimal an ensure to rings aluminium reagent. the Insert extraction RNA into immersing Adjust the sample volume before with RNA extraction reagent to thaw final volumeto 2 ml and vortex vigorously. sample allow not do • • • • Nanodrop andAgilent Bioanalyzer for qualityassessment (optional) 2 mlRNase-free tubes RNase-free water 70 %ethanol(Molecular Biology grade) Chloroform (Molecular Biology grade) Isopropanol (Molecular Biology grade) Liquid nitrogen orRNAlater solution(Ambion,Cat#7020)for tissue collection Tube holderpackfor extraction kits(Diagenode,CatNo.O-ring-15) Single Cycle Valve for Bioruptor Bioruptor RNA extraction kit(Diagenode,Cat.No.AL-001-0050) and homogenization Bioruptor @ diagenode.com /info Total sonication time Temperature Sonication cycle Power ® ® : Hposition(High) Water Cooler (Diagenode,Cat.No.BioAcc-Cool) disruption tissuefor UCD-300) UCD-200, No. Cat. (Diagenode, Plus Standardor ® fe ec cce vre smls n vsal cek the check visually and samples vortex cycle, each after : 4°C @ diagenode.com // : 30seconds ON,30seconds OFF : 1-3cycles ® North America -Diagenode Inc. Plus(Diagenode,Cat.No.VB-100-0001) without reflecting bars. reflecting ® Watercrushed BioAcc-Cool)or No. Cooler (Cat. /orders.na @

diagenode.com /info.na @ diagenode.com www.diagenode.com 2 Europe -Diagenodes.a. PROTOCOL 20. 19. RNA quantitation andqualityassessment 18. 17. 16. 15. 14. 13. 12. 11. 10. RNA isolation 9. If contamination of genomic DNA is expected, extract again by adding an equal volume of chloroform to the the to chloroform of volume equal an adding tube. by new again the to extract transferred expected, is phase DNA aqueous genomic of contamination If separation phase subsequent used. for be not important is should mixing alcohol isoamyl Thorough with mixed complex Chloroform nucleoproteins of dissociation complete the permits step This /orders Please note that optimization might be required depending on sample format (fresh or frozen tissue), tissue), frozen or degradation. (fresh RNA muscles. prevent like to format tissues chosen be sample fibrous on should with time occur may depending sonication required shortest disruption The be Incomplete amount. might tissue and type optimization tissue that note Please The table below shows suggested applications for RNA within different RIN ranges: ranges: RIN different the with within RNA for correlated be applications should and example). for suggested shows experiments microarray, or below table downstream (RT-PCR The run be desired to the on assay depends specific threshold values RIN Asess theintegrity ofRNAusingtheAgilent 2100Bioananlyzer (orBioRadExperionsystem). . Ratio OD260/230 greater than 1.8 is considered good. A low value might indicate organic contamination. ratio contamination. A organic indicate contamination. might value protein low A good. indicate might considered is 1.8 ratio than low A greater degradation. RNA good. OD260/230 indicate Ratio considered might 2.1 is than 1.8-2.0 greater OD260/280 Ratio the ensure to 260/230 purity ofRNA. OD and 260/280 OD ratio analyze and Nanodrop a using RNA Quantify Take analiquotfor quantitation andqualityanalysis. Store RNA-80°C. pipetting. Thesolutioncan beincubated at55-60C°for 10minif thepellet ishardby to dissolve.carefully resuspend yield), RNA expected on depending μl (100-300 water RNase-free Add dry thepellet. over- roomnot temperature.at Do min 5-10 forpellet the air-dry and supernatant the Remove Add 1mlof70%ethanol,vortex samples andcentrifuge for 10minat12.000g Remove thesupernatant andkeep thepellet. pellet gel-like A 4°C. at min forms onthesideandbottom ofthetube. 10 for g 12.000 at centrifuge and mix isopropanol, of ml 0.8 Add DNA- the aspirate to not care Take tube. containing white interface ml andorganic bluephase. 2 new a to phase upper colorless the Transfer Add 0.4mlofchloroform, vortex andcentrifuge at12.000gfor 10minat4°C. a new 2mlRNase-free tube. Centrifuge samples at 3.000 rpm for 5 min at room temperature and transfer the supernatant to extraction RNA of color blue the change may Vortex tubesvigorously after sonication andincubate for 5minatroom temperature. hemoglobin or iron blood, of amount reagent. high with Samples RNA. quality low prevent 7.0-10.0 4.1-6.9 1-4 RIN Value @ diagenode.com /info excellent to outstanding moderate to regular degraded to low RNA quality @ diagenode.com // North America -Diagenode Inc. Highly demanding genearray assays qRT-PCR applications PCR assays withshortregions ofamplification Suggested application /orders.na Do not sonicate longer than 3 cycles 3 than longer sonicate not Do @ diagenode.com /info.na @ diagenode.com www.diagenode.com to to 3 Europe -Diagenodes.a. PROTOCOL Left picture shows samples before sonication. Rightpicture shows disrupted sample. An example ofmousebrain disruptionusingRNAextraction tubesandRNAextraction reagent. Figure 1. Example oftissue disruption protocol andanalyzed on BioAnalyzer (Agilent). Bioruptor the with disrupted was Tissue samples. brain Total mouse RNAlater-treated 6-11) efficiently and (lanes fromRNA extracted 1-5) snap-frozen (lanes Figure 2. Examples oftotal RNAprofiles obtained from animaltissues /orders @ diagenode.com /info snap-frozen @ diagenode.com // ® tnad UD20. oa RA a etatd s ietd n the in directed as extracted was RNA Total (UCD-200). Standard RNAlater-stored North America -Diagenode Inc. /orders.na @ diagenode.com /info.na @ diagenode.com www.diagenode.com 4 Europe -Diagenodes.a. PROTOCOL /orders RIN 7.6 RIN 8.0 Muscle RIN 7.6 @ Brain Liver diagenode.com /info @ diagenode.com // North America -Diagenode Inc. /orders.na @ diagenode.com /info.na largely intact. is RNA the that indicating RNAs profiles all in small present are that Note (Agilent). analyzed BioAnalyzer on and the protocol (UCD- in described with as 300) Plus disrupted Bioruptor muscle Tissues were panel). skeletal (bottom (upper and brain panel), liver (middle panel) mouse from profiles RNA Total of pure RNAwithhighRIN. extraction Efficient Figure 3. @ diagenode.com www.diagenode.com 5 Europe -Diagenodes.a. PROTOCOL for complete tissue disruption using RNA extraction beads vs 15 cycles without RNA extraction beads. RNA beads. extracted fromextraction asample disrupted inthepresence ofRNAextractionRNA beadsshows significantly higherRQI. without cycles 15 vs requiredbeads areextraction cycles RNA 2 using only disruption that tissue extraction completeNote for RNA panel). (bottom the beads extraction and RNA Bioruptor with or using panel) liver (upper mouse without kit from obtained RNA total of traces (BioRad) Experion Pre-filled RNA extraction tubesimprove tissue disruptionandRNAquality. Figure 4. /orders @ diagenode.com /info RQI 7.8 RQI 5.7 @ diagenode.com // North America -Diagenode Inc. - + RNAextraction beads RNA extraction beads /orders.na @ diagenode.com /info.na @ diagenode.com www.diagenode.com 6 Europe -Diagenodes.a. PROTOCOL Troubleshooting guide xetd il o RA e mg per RNA of yield Expected Low yields Low ratio OD260/280 high to due baseline value RIN Low Degraded RNA of tissue ea el 6-15 1X10^6 cells cells HeLa g µ 2-5 Brain g µ 0.3-1 muscle Skeletal g µ 4-10 Liver /orders RIN 5.6 : @ diagenode.com /info Problem per µg @ diagenode.com // Sample manipulated too much completely RNA isnotsolubilized Protein contamination possible DNA/protein contamination is Sample sonicated too much reagent absence ofRNAextraction Frozen tissue thawed in Improper storage ofRNA stabilization before freezing orRNAlater North America -Diagenode Inc. Possible cause /orders.na rcs tsu immediately tissue Process dry vacuum drysample. to pellet completely.lyophilizeor allownot Do not Do hs wt te N sample (step 9intheprotocol) RNA the with phase Be sure not to carry any organic of RNAextraction reagent. ml 2 for mg 50 than more use not Do used. tissue much Too (see additionalprotocol) I DNase RNase-freeTreat with particulate chloroform addition before any material Remove 2 ml of RNA extraction reagent. Do not use more than 50 mg for the aqueousphase with DNA) (containsinterphase the of any take to not sure Be matter remains. sonicate particulate some if evenlonger not Do enough. are Avoid long sonication.1-2 cycles to RNAextraction reagent immediately tissue frozen Add freeze cycles Store RNA at -80°C avoid thaw- after dissection @ diagenode.com /info.na Suggested solution @ diagenode.com www.diagenode.com 7 Europe -Diagenodes.a. PROTOCOL Additional protocol for DNaseItreatment reagent ispowered byIsogen.Bioanalyzer isatrademark ofAgilent Technologies,extraction Inc.Experionisatrademark ofBio-Rad. RNA Diagenode SA. Diagenode of registeredtrademark a is Bioruptor owners. respective their or Diagenode of retrieval systems, or translated into any language or computer language. The trademarks mentioned herein are the property reproduced,reserved.be publicationrights may this All of SA. part Diagenode No 2012 © transmitted, transcribed,stored in RNA contains someDNA Low ratio OD260/230 • • • • • /orders Wash thepellet once with70%ethanol,air-dryandresuspend inRNase-free water of isopropanol to precipitate RNA Extract RNA samples with 100 Add EDTA to final concentration 2mM Incubate sample for 15-30minatroom temperature µl ofRNasin(optional)andRNase-free water to final volume of50µl 10 to up Combine @ diagenode.com /info g of RNA, 1 RNA, of µg @ diagenode.com // noul mtra ws not was removed before extraction material Insoluble of RNAextraction reagent ml 2 for used tissue much Too aqueous the phase was with interphase removed the of Part etc) (chloroform, polysaccharides Organic contamination µl of RNA extraction reagent and 20 l), 5 U/µl), (1 RNase-freeDNase of µl North America -Diagenode Inc. /orders.na eoe n particulate chloroform addition before any material Remove 2 ml of RNA extraction reagent. Do not use more than 50 mg for the aqueousphase with DNA) (containsinterphase the of any take to not sure Be sample (step 9intheprotocol) RNA the with phase Be sure not to carry any organic of RNAextraction reagent. ml 2 for mg 50 than more use not Do used. tissue much Too o ue n C ad qRT- (RNase-free) isrecommended and I DNase with PCR treatment PCR, in use For @ diagenode.com /info.na µl of chloroform. Use 80 l of 10X DNase buffer,1 DNase 10X of µl @ diagenode.com www.diagenode.com µl

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