Poly(A) Tail-Length Assay Kit - Thermo Fisher...
Transcript of Poly(A) Tail-Length Assay Kit - Thermo Fisher...
USB,thelogodesign,HotStart-ITandPrepEaseareregisteredtrademarksof Affymetrix,Inc.Poly(A)Tail-LengthAssayKit–Patentpending.HotStart-ITTaqDNAPolymerase–Patentpending.TestedUserFriendlyisatrademarkofAffymetrix,Inc.PrepEase products are covered under European Patent EP 0496822 and US Patent 6,428,703.The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular SystemsandF.Hoffmann-LaRocheLtd.SYBR is a registered trademark of Molecular Probes and is provided under an agreement with Molecular Probes.RNaseAway™isatrademarkofLifeTechnologies.RNaseZap®isaregisteredtrademarkofLifeTechnologies.
©2010Affymetrix,Inc.Allrightsreserved.
Affymetrix, Inc.26111 Miles RoadCleveland,Ohio44128USA P-76450Awww.usbweb.com rev 02/10
Poly(A) Tail-Length Assay Kit
Product Number 76450 5 G/I Tailing, 20 RT, and 400 PCR reactions
STORAGEStore at -20°C.
Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.
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CONTENTSComponents .....................................................................................................3
Quality Control .................................................................................................3
Safety Warnings and Precautions ...................................................................4
Introduction ......................................................................................................4
Assay Procedure Overview .............................................................................5
Materials Not Supplied ....................................................................................6
Protocol ............................................................................................................6
Reagent and Sample Handling ......................................................................6
Starting Materials ..........................................................................................7
Assay Controls ..............................................................................................7
Thermal Cycler Programs ..............................................................................8
Protocol ............................................................................................................8
G/I Tailing ......................................................................................................8
Reverse Transcription ....................................................................................9
PCRAmplification .......................................................................................10
Detection .....................................................................................................11
Supplementary Information ...........................................................................11
Data Analysis ..............................................................................................11
PCR Primer Design .....................................................................................13
Analysis by Gel Electrophoresis ..................................................................15
Troubleshooting .............................................................................................17
References .....................................................................................................18
Related Products ...........................................................................................18
Contact Information .......................................................................................19
COMPONENTSAll reagents have been extensively tested and carefully prepared to meet USB® standards. It is recommened that the reagents be used as directed in order to achieve the best possible results.
Thiskitcontainsreagentssufficientfor5G/Itailing,20reversetranscription,and400PCRreactions.Inaddition,thiskitincludesHeLatotalRNAandcontrolhuman actin PCR forward primer that can be used to verify components and protocol.
The following components are included with each kit:
5X Tail Buffer Mix 25 µl10X Tail Enzyme Mix 12 µl10X Tail Stop Solution 15 µl5X RT Buffer Mix (includes RT primer) 100 µl10X RT Enzyme Mix 50 µl5X PCR Buffer Mix 2 x 1.2 mlUniversalPCRReversePrimer,10μM 410μlHotStart-IT®TaqDNAPolymerase,1.25units/μl 2x250unitsControl,humanactinPCRForwardPrimer,10μM 8μlControl,HeLaTotalRNA,100ng/μl 10μlMgCl2,25mM 1mlWater,Nuclease-Free 8x1ml
Theenclosedreagentsshouldbestoredat-15°Cto-30°C(NOTinafrost-freefreezer).HeLatotalRNAshouldbestoredat-80°C.Afterthawingforuse,keepreagents on ice.
QUALITY CONTROLThePoly(A)Tail-LengthAssayKitisaTestedUserFriendly™productassuringreliable results. This kit is functionally tested for actin and k-ras poly(A) tail-lengthdetectionfromHeLatotalRNAfollowingtheprotocolinthemanual.AllcomponentsweretestedforcontaminatingssDNAanddsDNAendonucleases,ssDNAanddsDNAexonucleases,andribonucleases.Properlyhandledandstored components are guaranteed for optimal performance for at least 6 months from the date received.
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SAFETY WARNINGS AND PRECAUTIONSWarning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.
Caution:Allchemicalsshouldbeconsideredaspotentiallyhazardous.We,therefore,recommendthatthisproductishandledonlybythosepersonswhohave been trained in laboratory techniques and that it is used in accordance with theprinciplesofgoodlaboratorypractice.Wearsuitableprotectiveclothing,suchaslabcoat,safetyglasses,andgloves.Careshouldbetakentoavoidcontactwithskinandeyes.Inthecaseofcontactwithskinoreyes,washimmediatelywithwater.SeeMSDS(MaterialSafetyDataSheet)forspecificadvice.
INTRODUCTIONThepoly-adenylatedtail(poly(A)tail)onnearlyalleukaroyoticmRNAsplaysanumberofimportantrolesinmRNAmetabolismincludingenhancingtranslation,mRNAstabilityandtransportfromthenucleus(1,2). Studies in several model organismshaveshownregulateddeadenylationisratelimitingformRNAdegradation.Importantly,deadenylationisnowrecognizedasamechanismofmiRNAmediatedgeneregulation(3,4).Thus,identifyingchangesinpoly(A)tail-lengthcanyieldinsightsintomRNAregulationandsubsequentphysiologicalimpact.
ThePoly(A)Tail-LengthAssayKitusesfourkeystepstoenablepoly(A)tail-lengthdetermination.InStep1,poly(A)polymeraseaddsalimitednumberofguanosineandinosineresiduestothe3'-endsofpoly(A)-containingRNAs(5,6). InStep2,thetailed-RNAsareconvertedtocDNAthroughreversetranscriptionusingthenewlyaddedG/Itailsastheprimingsites.InStep3,PCRamplificationproductsaregeneratedusingtwoprimersets.Agene-specificforwardandreverse primer set designed upstream of the polyadenylation site (e.g. the 3'-UTR) is produced as a control for the gene-of-interest. The second set of primersusesthegene-specificforwardprimerandtheuniversalreverseprimerprovided with the kit to generate a product that includes the poly(A) tails of thegene-of-interest.Finally,inStep4,thePCRproductsareseparatedonanagarose or polyacrylamide gel. The poly(A) tail-lengths of the gene-of-interest arethesizesofpoly(A)PCR-amplifiedproductsminusthecalculatedlengthofthegene-specificforwardprimertotheputativepolyadenylationstartsite.
ASSAY PROCEDURE OvERvIEWThePoly(A)Tail-LengthAssayKitisdesignedforG/ItailinguptofivesamplesoftotalRNA.Allnecessarycomponentsareprovidedtoperform4reversetranscriptionand80PCRreactionsoneachofthefivetail-extendedsamples.Reaction products are then assessed by gel electrophoresis.
The protocol includes the following steps:
•Step1:G/ITailing (60minincubation) •Step2:ReverseTranscription (70minincubation) •Step3:PCRAmplification (30-60minincubation) •Step4:Detection
Figure 1. Poly(A) Tail-Length Assay Procedure.
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Starting MaterialsAtypicalassayreactionuses0.1–2μgoftotalRNA.TheamountoftotalRNArequiredperassaydependsonthetargetabundanceinthesample.ItisimportanttouseRNAthatiscompletelyfreeofcontaminatinggenomicDNA.ItisgenerallyunnecessarytotreattheRNAwithDNaseItoremoveanygenomicDNAcontamination.However,certainRNApreparationsmayyieldnon-specificamplificationproductsthatcanberemovedbytreatingtheisolatedRNAwithrDNaseI(PN78311).SamplestreatedwithDNaseIshouldbeextractedwithphenol-chloroformorpurifiedwithacolumn-basedprocedure.
Assay ControlsPreparean“AssayPositiveControl”byusingthesuppliedHeLaTotalRNAandhumanactinPCRForwardPrimer.Thiscontrolwillbeusedtoassessassaycomponents and procedures.
Preparea“NoRTNegativeControl”toassessnon-specificamplificationbysubstitutingthe10XRTEnzymeMixwithNuclease-FreeWater.
Preparea“SpecificPrimerControl”toassessspecificityofthegene-specificPCR forward primer by substituting the Universal PCR Reverse Primer with the gene-specificPCRreverseprimer(notsupplied).
The following table summarizes the recommended reactions that should be performed.
Step G/I TailingReverse
TranscriptionPCR Amplification
Tail PCR Primers Specific PCR Primers
Input Enzyme Buffer Enzyme Buffer Forward Reverse Forward Reverse
Assay Positive Control
3HeLaRNA 3 3 3 3 3actin 3Universal n/a n/a
Sample
No RT Negative Control
test RNA
3 3 water 3 S 3Universal n/a n/a
Specific Primer Control
test RNA
3 3 3 3 n/a n/a S S
Poly(A) Testtest RNA
3 3 3 3 S 3Universal n/a n/a
3 indicates use of supplied components.Sindicatesgene-specific.
MATERIALS NOT SUPPLIEDThe following materials are required for use with the kit:
•100ngto2μgoftotalRNA(seeStartingMaterialsandRelatedProductsections for advice and sample preparation kits)
•SpecificPCRforwardandreverseprimersdesignedforthegene-of-interest(see Supplementary Information for design guidelines)
•Microcentrifuge•Thermalcycler•Adjustableprecisionpipettes•RNase-freefilterpipettetipsandNuclease-freetubes•AppropriatePCRplates/tubesforinstrument•Disposablegloves•Gelelectrophoresis
-Molecularweightmarker(USBPN76712or76710)-DNAloadingbuffer(USBPN76715or76720)-2-2.5%agarose(USBPN32802)gelandTAEbuffer(USBPN75904or
74015)-4-6%non-denaturingpolyacrylamide(USBPN75848)andTBEbuffer(USBPN75891)
-UVtransilluminatororfluorescenceimagescanner
PROTOCOLReagent and Sample HandlingThawreagentsonice,mixthoroughlybeforeuseandimmediatelyreturnunusedmaterialsto-20°C.Whenpreparingworkingreagents,measurecomponentsaccurately,mixthoroughly,spinbrieflyandkeeponice.Assemblereactionsonice or at the indicated temperature throughout the procedure.
WhenworkingwithRNA,wearglovesatalltimeswhilehandlingreagents,materialsandequipmenttopreventRNasecontaminationfromhands.CleanpipettesandworkareaswithRNaseAway™orRNaseZap® to reduce the risk of RNasecontamination.UseRNase-freeplasticwareandRNase-freebuffersandreagents.
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Thermal Cycler ProgramsDuringthePoly(A)Tail-LengthAssay,thesamplesareplacedinathermalcyclerthreetimes.Therefore,werecommendprogrammingyourthermalcycler(s)withthe following programs prior to sample processing.
Programs 1. G/I Tailing: 37°C for 60 min 2. Reverse Transcription: 44°C for 60 min; 92°C for 10 min; and 4°C hold 3.PCRAmplification:
Two-StepPCR,Recommended Three-Step PCR
94°C for 2 min 30-35 cycles of: 94°C for 10 sec 60°C for 30-60 sec72°C for 5 min 4°C hold
94°C for 2 min30-35 cycles of: 94°C for 10 sec 58°C for 30 sec 72°C for 30 sec72°C for 5 min4°C hold
Note: Certain targets may exhibit sub-optimal amplification with the Two-Step PCR protocol. The Three-Step PCR protocol should be used in cases where weak PCR amplification is observed.
PROTOCOLStep 1: G/I TailingUsethefollowingprotocoltoaddpoly(G/I)tailstoatotalRNAsample.Forthepositivecontrol,substitutetheprovidedHeLatotalRNAforanexperimentalsample. This standard protocol applies to a single 20 µl G/I Tailing reaction.
1. Thaw frozen reagents on ice and mix thoroughly by vortexing. Enzyme mixes shouldbegentlyflickedtomix.Centrifugebriefly.
2. Add the following reagents in Table 1 to a nuclease-free tube. Mix gently bypipettingupanddownandthencentrifugethetubebrieflytocollectthecontents.Keepsamplesonice.
Table 1. G/I Tailing MixReagent Per reactionTotalRNAsample,1μg(0.1–2μg) up to 14 µl5X Tail Buffer Mix 4 µl10X Tail Enzyme Mix 2 µlWater,Nuclease-Free to 20 µl
3. Incubate at 37°C for 60 min
4. Add 2 µl 10X Tail Stop Solution and mix well.
5. Proceed to Step 2: Reverse Transcription. Alternatively, tailed RNA samples can be stored at -20°C until ready to proceed to Step 2.
Step 2: Reverse TranscriptionUsethefollowingprotocoltoreversetranscribethepoly(G/I)tailedRNA.Thisstandard protocol applies to a single 20 µl reverse transcription reaction. Master mixes for multiple reactions can be made by increasing the volumes of reaction components proportionally.
1. Thaw frozen reagents on ice and mix thoroughly by vortexing. Enzyme mixes shouldbegentlyflickedtomix.Centrifugebriefly.
2. Add the following reagents in Table 2 to a nuclease-free tube. Mix gently and brieflyspindownthetubecontents.Keeponice.
Table 2. RT MixReagent RT + RT - (control)G/ITailedRNASample 5 µl 5 µl5X RT Buffer Mix 4 µl 4 µl10X RT Enzyme Mix 2 µl -Water,Nuclease-Free 9 µl 11 µl
Note:Eachkitsupports20x20μlreactions.
3. Incubate at 44°C for 60 min; 92°C for 10 min; and at 4°C hold.
4.ProceedtoStep3:PCRAmplification.Alternatively, cDNA samples can be stored at -20°C until ready to proceed to Step 3.
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Step 3: PCR AmplificationUsethefollowingprotocoltoPCRamplifythepoly(G/I)tailedcDNA.Thisstandard protocol applies to a single 25 µl PCR reaction. Master mixes for multiple reactions can be made by increasing the volumes of reaction components proportionally.
1.DiluteeachRTsamplebyadding20μlNuclease-FreeWater(40μlfinalvolume).
2. Thaw frozen reagents on ice and mix thoroughly by vortexing. Mix HotStart-IT® TaqDNAPolymerasebygentlyflicking.Centrifugebriefly.
3. Add the following reagents in Table 3 to a nuclease-free tube. Mix gently and brieflyspindownthetubecontents.Keeponice.
Table 3. PCR Mix
ReagentRT +Tail PCR
RT -Tail PCR
RT +Specific
PCR
RT -Specific
PCRDiluted RT sample up to 5 µl up to 5 µl up to 5 µl up to 5 µl5X PCR Buffer Mix 5 µl 5 µl 5 µl 5 µl10μMGene-SpecificPCRForwardPrimer
1 µl 1 µl 1 µl 1 µl
10 µM Universal PCR Reverse Primer
1 µl 1 µl - -
10μMGene-SpecificPCRReverse Primer
- - 1 µl 1 µl
25mM MgCl2* optional optional optional optional1.25 units/µl HotStart-IT® Taq DNAPolymerase
1 µl 1 µl 1 µl 1 µl
Water,Nuclease-Free to 25 µl to 25 µl to 25 µl to 25 µl
*Additional MgCl2mayberequiredtoincreaseamplificationefficiencyofcertaintargets and is provided in this kit.
4. Proceed to Step 4: Detection. Alternatively, PCR products can be stored at -20°C until ready to proceed to Step 4.
Figure 2. Example of poly(A) tail-length determination. A-tail length is (z-y-35) where z can vary based on gel results.
Step 4: DetectionThe size of PCR products can be assessed by running an aliquot of the reaction onanagaroseorpolyacrylamidegel.Tostart,werecommendloadingonehalfofeachPCRreaction(12.5μl)perlaneona2.5%agaroseTAEgel.Forincreasedresolution,loadonehalfofeachPCRreaction(12.5μl)perlaneona5% non-denaturing polyacrylamide TBE gel. Stain gels with ethidium bromide or SYBR® Gold and visualize with a standard ultraviolet transilluminator or fluorescenceimagescanner.
See the Supplementary Information Section for guidelines on gel electrophoresis and data analysis.
SUPPLEMENTARY INFORMATIONData AnalysisThePoly(A)Tail-LengthAssayKitdeterminesthelengthdistributionofmRNApoly(A)tails.PCRproductsofmRNAswithshorttailswillyielddiscretebands,whereasmRNAswithlongtailswillyieldasmearonthegel(Fig.1).PCRamplificationwiththegene-specificforwardprimerandUniversalreverseprimeramplifiesthesequenceupstreamofthepolyadenylationstartsitesite(e.g. the 3'-UTR) to the end of the poly(A) tails. The poly(A) tail-lengths of the gene-of-interestarethesizesofpoly(A)PCR-amplifiedproductsminusthecalculatedlengthofthegene-specificforwardprimertotheputativepolyadenylationstartsite(Fig.2).PCRwiththegene-specificforwardandreverseprimersshould amplify only the upstream sequence of the expected size to validate the specificityofthegene-specificforwardprimer.The“NoRTNegativeControl”reactionshouldhavenosignal.ExamplesofresultsareshowninFigs.3and4.
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Figure 3. Comparison of human actin poly(A) tail-lengths in brain, muscle, liver and HeLa cell.OnemicrogramtotalRNAand4μlofdilutedRTsampleswereusedinG/ITailingandPCRreactions,respectively.Therecommendedtwo-stepPCRprogramwasused.OnehalfofeachPCRreaction(12.5μl)wasanalyzedon6%non-denaturingpolyacrylamide-TBE gel stained with SYBR® Gold (A),and2.5%agarose-TAEgelstainedwith ethidium bromide (B).RT(+);NoRTNegativeControl(-);poly(A)tailPCR(A);gene-specificPCR(S);and100bpDNALadder(USBPN76712)(M).
Figure 4. Detection sensitivity of the USB Poly(A) Tail-Length Assay. Actin poly(A) tail-lengthwasdeterminedfromatwo-foldserialdilutionHeLatotalRNA.SampleswereprocessedasdescribedinFig.3B(A).Thetopimagewasquantifiedbydensitometry(B).
PCR Primer DesignUniversal reverse primer: The Universal PCR Reverse Primer supplied with each kit is used as the reverse primer in poly(A) tail-length detection PCR reactions. It issuppliedat10μMandusedatafinalconcentrationof400nM.
Gene-specificforwardandreverseprimers:Thesearetheprimersthatareuser-definedforthegene-of-interest.Theyshouldbedilutedto10μMinTEBuffer(PN75893)andusedatafinalconcentrationof400nM.Theforwardprimerisused with the universal reverse primer to generate the poly(A) tail-length PCR productsandthegene-specificforwardandreverseprimersareusedtogethertoverifythespecificityoftheforwardprimerandthepresenceofthetargetwithintheRNAsample.
Thegene-specificPCRprimersshouldbelocatedwithin50-300nucleotidesupstream of the poly(A) start site to allow proper resolution of PCR products bygelelectrophoresis.Ifpossible,thegene-specificreverseprimershouldbe located immediately upstream of the poly(A) start site for straightforward calculation of the poly(A) tail-lengths. We recommend using computer programs designed to select appropriate primers in a given sequence. Several public primer databases are available on the internet. Some examples of databases include:
NCBI,http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
Primer3,http://frodo.wi.mit.eduIDT,http://www.idtdna.com/Scitools/Applications/Primerquest
Ingeneral,followtheseguidelinesforbestresults: •Primersshouldrangeinlengthfrom19to30nucleotides, •G+Ccontentintherangeof30to50%, •Tmvaluesrangingfrom55-60°C, •Analyzeforcross-reactivityintheorganism’sdatabase.
DuetotheAT-richcontentin3'UTRsequences,itmaybedifficultinsomecasestodesignaprimerthatfitsthesespecifications.Wehavealsotestedthatprimers with Tm below 55°C and have found that these can work in this assay as longasthegene-specificforwardprimerhasbeenvalidatedforspecificprimingandamplificationofthegene-of-interest.Ingeneral,twospecificforwardprimersandonespecificreverseprimershouldbedesignedpergene-of-interestforbestpossibleresults.Anexampleofusingdifferentspecificforwardprimerdesignsforpoly(A)tail-lengthdeterminationisshowninFig.5.
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Ck-ras primer 5' → 3' Sequence Tm (°C) GC (%) Length(nt)
k1 CCACAGAGCTAACTGGGTTACAGT 58.4 50 24 k2 TGTAACATGTTTACCTGGAATGT 52.3 35 23 k3 TGTATAGTGTAAACTGAAACATGCAC 53.6 35 26 k4 CATTGTGCTTTCTTTTGTGGGACA 56.5 42 24 k5 TGGTTGCGCTGACCTAGGAATGTT 60.8 50 24 k6 CGCTGACCTAGGAATGTTGG 55.6 55 20 k-RP GTCACTGTAACTATTTTTATTAC 45.2 26 23
Figure 5. Different gene-specific forward primer designs for poly(A) tail-length determination of k-ras from HeLa total RNA. Primer location on k-ras transcript (B) and primer information are shown (C).SampleswereprocessedasdescribedinFig.3B(A).NoRTNegativeControl(RT-);poly(A)tailPCR(A);gene-specificPCR(S);and100bpDNALadder(USBPN76712)(M).
Analysis by Gel ElectrophoresisPreparing and running agarose gels1. Chooseahorizontalgelelectrophoresisapparatuswithacapacityof≥15μl
per well.
2. Prepare2.5%agaroseTAEgelbymixing2.5gmagarose(PN32802)per 100 ml 1X TAE Buffer (e.g.PN75904or74015,dilutedto1Xwithdistilledwater).
3. Heat to boil the agarose until completely dissolved.
4. Coolto~65°C,thenaddethidiumbromideto1μg/ml(or1dropofethidiumbromide,PN75816,per100ml).
5. Pour the gel solution into the gel tray with comb to form wells and let set completely.
6. Prepare sample by adding loading buffer to 1X (e.g.4μlof6XLoadingBuffer,PN76715orPN76720).
7. Mix and quick spin to collect tube contents at the bottom of the tubes.
8. Load14μlofthedye-PCRmixsampleperlane.Forthefirstandthelastlane,loadDNAmarker(e.g.3μlof100bpDNALadder,PN76712).
9. Run in 1X TAE Buffer (e.g.PN75904,dilutedto1Xwithdistilledwater)at 150 volts for 40-60 min.
10. Visualize and document with a standard ultraviolet transilluminator or fluorescenceimagescanner.
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TROUBLESHOOTINGProblem Possible causes and solutionsWeak or no signal 1.PoorRNAsamplequality – CheckRNAintegritybygelelectrophoresis
or bioanalyzer. 2.LowabundantRNAtarget – IncreasetheamountoftotalRNAto2μgper
G/I Tailing reaction. – Usepoly(A)-enrichedRNA.Upto0.5μg
poly(A)RNAsampleperreactioncanbeused.
– Increase the sample volume for gel analysis. 3. Sub-optimal PCR condition – Increase the amount of diluted RT to 5 µl per
PCR reaction. – OptimizeMgCl2 for the PCR reaction. – Try different PCR forward primer. – Increase the number of PCR cycles. – Decrease PCR annealing temperature. – Increase PCR extension time. – Try the Three-Step PCR protocol. – Use the supplied PCR reagents. These
components have been optimized for use with this assay.
Non-specific signal 1.PoorRNAsamplequality – This may indicate the presence of
contaminatinggenomicDNAintheRNAsample.TreattheRNAsamplewithDNaseIandremovetheDNaseIbyphenol-chloroform extraction or a column-based purification.
2. Isoform detection – Check if the gene-of-interest has different
isoforms and the unexpected signals correspond to the presence of alternatively spliced transcripts.
– Designnewspecificforwardprimersthatallow isoform discrimination.
Preparing and running polyacrylamide gels1.Chooseaverticalgelelectrophoresisapparatuswithacapacityof≥15μlperwell.Followthemanufacturer’sinstructionsforthedetailsofassemblinggelapparatus.
2.One10cmx15cmx1mmgelrequires15mlofgelsolution.Prepare5%polyacrylamide TBE gel by mixing the following:
For 15 ml
5XTBE(PN75891) 3 ml
40%acrylamidesolution(19:1acrylamide:bis-acrylamide,PN75848)
1.9 ml
water to 15 ml 10.1 ml
Add the following reagents immediately before pouring the gel:10%ammoniumpersulfate(PN76322)inwaterTEMED(PN76320)
120 µl16 µl
3. Pour the gel solution into the gel cassette and place comb to form wells and let polymerize completely at room temperature for at least 30 min.
4. Prepare sample by adding loading buffer to 1X (e.g.4μlof6XLoadingBuffer,PN76715PN76720).
5. Mix and quick spin to collect tube contents at the bottom of the tubes.
6.Load14μlofthedye-PCRmixsampleperlane.Forthefirstandthelastlane,loadDNAmarker(e.g.3μlofDNALadder,100bp,USBPN76712).
7. Run in 1X TBE Buffer (e.g.PN75891,dilutedto1Xwithdistilledwater)at ~7watt,constantpoweror~25mAmp,constantcurrentfor30-60min.
8. Stain with SYBR®GoldNucleicAcidGelStain(LifeTechnologies)accordingtothemanufacturer’sinstructions.
9. Visualize and document with a standard ultraviolet transilluminator or fluorescenceimagescanner.
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Problem Possible causes and solutions 3. Sub-optimal PCR condition – Use the recommended Two-Step PCR
protocol. – Decrease the number of PCR cycles. – Designnewspecificprimers. – Use the supplied PCR reagents. These
components have been optimized for use with this assay.
4.DNAcontaminationduringsampleprocessing – Usefilter-barriertipsforassayset-up. – Replace all reagents for PCR.
If problems persist please contact Technical Support for assistance at (800)321-9322orUSBtechsupport@affymetrix.com.FortechnicalsupportoutsidetheU.S.,pleasevisitourwebsiteforup-to-datecontactinformationonthe USB product distributor within your area.
REFERENCES1.ParkerR.,andSongH.(2004)Nat Struct Mol Biol. 11,121-127.
2.AndersenK.R.,JensenT.H.,andBrodersenD.E.(2008)Biochim Biophys Acta. 1779,532-537.
3.WuL.,FanJ.,andBelascoJ.G.(2006)Proc Natl Acad Sci USA. 103,4034-4039.
4.EulalioA.,HuntzingerE.,NishiharaT.,RehwinkelJ.,FauserM.,andIzaurraldeE. (2009) RNA 15,21-32.
5.MartinG.,andKellerW.(1998)RNA 4,226-230.
6.KusovY.Y.,ShatirishviliG.,DzagurovG.,andGauss-MüllerV.(2001)Nucleic Acids Res. 29,E57-7.
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hlor
ide
7447
-40-
7 ~
2.8%
—
S:2
3 D
o no
t bre
athe
vap
our.
S:2
4/25
Avo
id c
onta
ct w
ith s
kin
Fo
rCom
ponent71195
:
and
eyes
.
Tr
is-H
Cl
1185
-53-
1 >
1%
—
S:3
6/37
Wea
r su
itabl
e pr
otec
tive
P
otas
sium
Chl
orid
e 74
47-4
0-7
> 1
%
—
cl
othi
ng a
nd g
love
s.
ForCom
ponent76465
:
P
otas
sium
Chl
orid
e 74
47-4
0-7
~1.
9%
S
ee in
form
atio
n ab
ove.
ForCom
ponents
7646
1 an
d
R:36/37/38Irritatingtoeyes,
7646
4:
See
re
spira
tory
sys
tem
and
ski
n.
Gly
cero
l 56
-81-
5 ~
50%
“R
egul
ator
y
S:2
6 In
cas
e of
con
tact
with
Inform
ation”
eyes,rinseim
mediatelywith
S
ectio
n pl
enty
of w
ater
and
see
k m
edic
al
ForCom
ponent76460
:
advi
ce.
Tris
-HC
l 11
85-5
3-1
~1.
6%
—
S
:36/
37 W
ear
suita
ble
prot
ectiv
e
Gly
cero
l 56
-81-
5 ~
25%
—
clot
hing
and
glo
ves.
ForCom
ponent75788:
Noapplicableinform
ation.
HeLaTotalR
NA
N/A
~100%
—
HA
ZA
RD
S ID
EN
TIF
ICAT
ION
C
HIP
B
ioha
zard
; Irr
itant
H
CS
B
ioha
zard
; Irr
itant
FIR
ST-
AID
ME
AS
UR
ES
EYES:Flushwithwaterfo
r15min.S
eekmedicaladviceifirritationpersists.
SKIN:Flushwithwater,thenwashthoroughlywithsoapandwater.R
emovecontam
inatedclothingandwash
befo
re re
use.
See
k m
edic
al a
tten
tion
if irr
itatio
n pe
rsis
ts.
INHALATION:R
emovethevictimfrom
exposureandmovetofreshair.Ifbreathingisdifficult,giveoxygen.Ifnot
breathing,giveartificialrespiration.Keepvictimquietand
warm.S
eekimmediatemedicalattention.
INGESTION:D
rinkwaterand
seekimmediatemedicalattention.Avoidalcoholicbeverages.N
evergiveanything
by m
outh
to a
n un
cons
ciou
s pe
rson
.
FIR
E-F
IGH
TIN
G IN
FOR
MAT
ION
Usemediasuitabletoextinguishthesupp
ortingorsurroundingfire.W
earNIOSH(orequivalent)app
rovedself
containedbreathingapp
aratus.Forsmallfiresonly:usecarbo
ndioxide,drypow
derorfo
am.E
mitsto
xicfumes
underfirecond
itions.ForGlycerol:Contactwithstrongoxidizingagentsmayprodu
ceanexplosion.
ExplosionLimitsfo
rGlycerol=Low
er-1.1;U
pper-Notavailable.
Flashpo
intforGlycerol=193°C(379.4°F);Autoignitiontemperaturefo
rGlycerol=400°C(752°F).
AC
CID
EN
TAL
RE
LEA
SE
ME
AS
UR
ES
C
autio
n: C
atal
og#
7578
8 is
isol
ated
from
hum
an s
ourc
es. H
andl
e al
l pro
duct
s pr
epar
ed fr
om h
uman
sou
rces
asifth
eywerecapableoftransm
ittinginfectiousagents.Avoidaccidentalinoculation,intravenousinjectionor
contactw
ithopenwound
s.W
ashthoroughlyafterhandling.Observeuniversalprecautionswhenworkingwith
thisprodu
ct.W
earapprop
riatepersonalprotectiveequipm
entand
clothinginclud
inglabcoat,safetyglasses,
glovesand
NIOSH-app
roved(orequivalent)respiratorapprop
riatefo
rthehazard.C
ontainth
espillwithaninert
abso
rben
t and
pla
ce in
a s
uita
ble
was
te c
onta
iner
. Avo
id c
onta
ct o
f mat
eria
l with
ski
n or
eye
s. U
se a
dequ
ate
vent
ilatio
n.
HA
ND
LIN
G A
ND
ST
OR
AG
E
Cau
tion:
Cat
alog
# 75
788
is is
olat
ed fr
om h
uman
sou
rces
. Han
dle
all p
rodu
cts
prep
ared
from
hum
an s
ourc
es
asifth
eywerecapableoftransm
ittinginfectiousagents.Avoidaccidentalinoculation,intravenousinjectionor
contactw
ithopenwound
s.W
ashthoroughlyafterhandling.Observeuniversalprecautionswhenworkingwith
thisprodu
ct.W
earapprop
riatepersonalprotectiveequipm
entand
clothinginclud
inglabcoat,safetyglasses,
glovesand
NIOSH-app
roved(orequivalent)respirator.Aqualifiedindu
strialhygienistshouldevaluateth
eneed
fo
r re
spira
tory
pro
tect
ion.
Use
ade
quat
e ve
ntila
tion.
Avo
id c
onta
ct o
f mat
eria
l with
ski
n or
eye
s. S
tore
kit
at -
20°C
aw
ay fr
om in
com
patib
le m
ater
ials
.
PE
RS
ON
AL
PR
OT
EC
TIO
N
Cau
tion:
Cat
alog
# 75
788
is is
olat
ed fr
om h
uman
sou
rces
. Han
dle
all p
rodu
cts
prep
ared
from
hum
an s
ourc
es
asifth
eywerecapableoftransm
ittinginfectiousagents.Avoidaccidentalinoculation,intravenousinjectionor
contactw
ithopenwound
s.W
ashthoroughlyafterhandling.Observeuniversalprecautionswhenworkingwith
thisprodu
ct.W
earapprop
riatepersonalprotectiveequipm
entand
clothinginclud
inglabcoat,safetyglasses,
glovesand
NIOSH-app
rovedrespirator.Aqualifiedindu
strialhygienistshouldevaluatetheneedfo
rrespiratory
protection.UserespiratoryprotectionapprovedbyNIOSH(orequivalent)and
app
ropriateto
thehazard.A
void
cont
act o
f mat
eria
l with
ski
n or
eye
s. M
echa
nica
l ven
tilat
ion
or lo
cal e
xhau
st a
s ne
eded
to c
ontr
ol e
xpos
ure
to
dust,vaporsormists.A
ccesstoasafetyshow
erand
eye-w
ash.
22 23
PH
YS
ICA
L A
ND
CH
EM
ICA
L App
earance:Kitcontainingvialsofsolutions
BoilingPoint:N
odataavailable
PR
OP
ER
TIE
S
Vapo
rPressure:Nodataavailable
Vapo
rDensity:N
odataavailable
Solub
ility(W
ater):Allcompo
nentsaresoluble
SpecificGravity:N
odataavailable
PercentVolatile:N
odataavailable
Evapo
rationRate:Nodataavailable
ChemicalFormula:Notapp
licable
STA
BIL
ITY
AN
D R
EA
CT
IvIT
Y
Pro
duct
is s
tabl
e un
der
norm
al c
ondi
tions
. Avo
id p
rolo
nged
exc
essi
ve h
eat w
hich
may
cau
se d
ecom
posi
tion.
Storeawayfrom
strongbases,strongacids,and
strongoxidizingagents.H
azardo
usdecom
positionprod
ucts
mayinclud
ecarbonoxides.Hazardo
uspolym
erizationwillnotoccur.ForGlycerol:Avoidstrongoxidizingagents
includ
ingmixtureswithhydrogenperoxide,p
otassium
permanganate,trifluorobrom
ide,calcium
hypochlorite,
nitricacid,sulfuricacid,perchloricacidandleadoxide.C
ontactwithSod
iumHypochloriteand
Hypochlorous
acid
may
cau
se a
n ex
plos
ion.
TO
XIC
OLO
GIC
AL
INFO
RM
ATIO
N
EFF
ECTS
OFOVEREXPOSURE:
EY
ES
: Con
tact
may
cau
se ir
ritat
ion.
SKIN:C
ontactmaycauseredn
ess,swellingandpainatanysite,especiallymucousmem
branes.
INHALATION:E
xcessiveinhalationofvapormaycauseirritation,coughand
shortnessofb
reath.
INGESTION:Ingestionorexcessiveexposuremayleadto
nausea,vom
itingand
diarrhea.Largeamountsmay
causeweakness,collapseandcoma.
TARGETORGANS:E
yesandSkin.
ADDITIONALINFO
RMATION:
Tris-HCl-RTE
CS:N
odataavailable.
Potassium
Chloride:Irritation,mutationandtoxicitydatalistedinRTE
CSund
erTS8050000.
Irritationdata:E
yeRabbit5
00mg/24H=M
ild(1972).
Toxicitydata:OralR
atLD50=2600mg/kg(1972).
Labo
ratoryexperimentshaveresultedinmutageniceffects.
Glycerol:Irritation,mutation,reprodu
ctiveeffectsandtoxicitydatalistedinRTE
CSund
erM
A8050000.
Irritationdata:E
yeRabbit5
00mg/24H=M
ild(1986).S
kinRabbit5
00mg/24H=M
ild(1986).
Toxicitydata:OralR
atLD50=12600mg/kg(1945).InhalationRatLC50=>570mg/m3/1H
(1970).
CAUTION:C
atalog
#75788isisolatedfrom
hum
ansources.H
andleallprodu
ctspreparedfrom
hum
ansources
asifth
eywerecapableoftransm
ittinginfectiousagents.Avoidaccidentalinoculation,intravenousinjectionor
cont
act w
ith o
pen
wou
nds.
Wea
r ap
prop
riate
per
sona
l pro
tect
ive
equi
pmen
t. W
ash
thor
ough
ly a
fter
han
dlin
g.
Observeuniversalprecautionswhenworkingwithth
isprodu
ct.
Definition(s):RTE
CS=RegistryofToxicEffectsofChemicalSub
stances.
ACGIH=AmericanConferenceofGovernm
entalInd
ustrialH
ygienists.
OSHA=Occup
ationalS
afetyandHealthAdm
inistration.
EC
OLO
GIC
AL
INFO
RM
ATIO
N
Noinform
ationavailable.
DIS
PO
SA
L C
ON
SID
ER
ATIO
NS
Dispo
seofm
aterialinaccordancewithapp
licablelocal,state,and
federalregulations.
TR
AN
SP
OR
TAT
ION
INFO
RM
ATIO
N
USDOT/IATA:N
oapplicableinform
ation.
RE
GU
LAT
OR
Y IN
FOR
MAT
ION
RCRA-Noapplicableinform
ation.
SARA302-Noapplicableinform
ation.
SARA313-Noapplicableinform
ation.
EPA
TSCASection8(b)-ForGlycerol,Tris-HCl,andPotassium
Chloride:ChemicalInventory.
ExposureLimits-ForGlycerol:ACGIHTLV-TWA10mg/m3(to
talparticulate).
OSHAPELTW
A:15mg/m3(to
taldust).
CaliforniaPropo
sition65-Noapplicableinform
ation.
This
dat
a sh
eet i
s b
ased
up
on in
form
atio
n be
lieve
d to
be
relia
ble.
The
Com
pany
mak
es n
o st
atem
ent o
r w
arra
nty
as to
the
accu
racy
or
com
plet
enes
s of
theinform
ationcontainedhereinwhichisofferedforyourconsideration,investigationandverification.Anyuseoftheinform
ationcontainedinth
isdata
shee
t mus
t be
det
erm
ined
by
the
user
to b
e in
acc
orda
nce
with
app
ropr
iate
app
licab
le re
gula
tions
.