Poly(A) Tail-Length Assay Kit - Thermo Fisher Scientific · PDF fileAll reagents have been...

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.

2 3

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.

4 5

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.

6 7

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.

8 9

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.

10 11

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.

12 13

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.

14 15

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.

16 17

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.

18 19

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.

RELATED PRODUCTSProduct Application Pack size Product numberPrepEase®RNAClean-UpKit Clean-upofRNA 10preps 78875 50 preps 78876 250 preps 78877AgaroseLE Gelelectrophoresis 25gm 32802 100 gm 250 gm 500 gm 1 kg

Product Application Pack size Product numberAmmonium Persulfate Gel electrophoresis 100 gm 76322DNALadder,100bp Gelelectrophoresis 500μl 76712DNALoadingBuffer,6X Gelelectrophoresis 1ml 76715 (included with 76710 and 76712) 5 ml6XDNALoadingBuffer(BXF) Gelelectrophoresis 1ml 76720 5 mlEthidium Bromide Drops Gel electrophoresis 5 ml 75816PCRMarkers,50-2,000bp Gelelectrophoresis 250μl 76710RapidGel,40%Liquid Gelelectrophoresis 500ml 75848 Acrylamide Stock SolutionTAEBuffer,10XSolution Gelelectrophoresis 1L 75904 5LTAEBuffer,50XSolution Gelelectrophoresis 100ml 74015TBEBuffer,5XSolution Gelelectrophoresis 1L 75891 5LTEMED Gel electrophoresis 100 ml 76320PrepEase®mRNAMiniSpinKit IsolationofmRNA 12preps 78878PrepEase®RNASpinKit IsolationofRNA 10preps 78765 50 preps 78766 250 preps 78767 PrepEase®RNASVESpinKit IsolationofRNA 10preps 78772 50 preps 78773 250 preps 78774PrepEase®PlantRNASpinKit IsolationofRNAfromplant 20preps 78770 cells 50 preps 78771PrepEase®RNA/ProteinSpinKit IsolationofRNA/protein 10preps 78870 50 preps 78871 250 preps 78872rDNaseI,RNase-Free Removalofcontaminating 1,000units 78411 DNA 2,500unitsTEBuffer,1XSolution Resuspension/dilutionof 100ml 75893 DNA 10x1ml 500 ml

Affymetrix, Inc.USB® ProductsUSA EuropeCleveland,Ohio Staufen,Germany(800) 321-9322 +49(0)76 33 - 933 400www.usbweb.com www.usbweb.de

USB products distributed outside the USA:Please visit the USB website at www.usbweb.com for up-to-date contact information within your area.

20 21

Mat

eria

l Saf

ety

Dat

a S

heet

Rev

isio

n: 0

4/13

/200

9

Haz

ard

info

rmat

ion

is p

rovi

ded

for

com

plia

nce

with

bot

h th

e

UKChemicals(HazardInform

ationandPackaging

)(CHIP)

Reg

ulat

ions

and

the

US

Haz

ard

Com

mun

icat

ion

Sta

ndar

d (H

CS

)

IDE

NT

IFIC

ATIO

N O

F T

HE

P

RO

DU

CT

NA

ME

:

PR

OD

UC

T C

OD

E:

EE

C N

UM

BE

R:

SU

BS

TAN

CE

/PR

EPA

RAT

ION

Poly(A)Tail-Leng

thAssayKit

76450

Non

e A

ND

CO

MPA

NY

SUPPLIER:

EMERGENCYCONTA

CT:

US

B® P

rod

ucts

- A

ffym

etri

x, In

c.

Che

mtr

ec: (

800)

424

-930

0

2611

1 M

iles

Ro

ad, C

leve

land

, Ohi

o 4

4128

Pho

ne: (

216)

765

-500

0

Out

sid

e U

SA

& C

anad

a: 7

03-5

27-3

887

P

leas

e vi

sit

our

web

site

at

ww

w.u

sbw

eb.c

om

for

cont

act

info

rmat

ion

on U

SB

pro

duc

t d

istr

ibut

ors

with

in y

our

area

.

CO

MP

OS

ITIO

N/

HA

ZA

RD

OU

S C

OM

PO

NE

NT

S

HA

ZA

RD

C

AS

NO

. %

WT

T

Lv

CH

IP R

& S

Phr

ases

ForCom

ponent7

6463

:

R:36/37/38Irritatingtoeyes,

Tris

-HC

l 11

85-5

3-1

~3.

9%

—

re

spira

tory

sys

tem

and

ski

n.

Pot

assi

um C

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



contactw

ithopenwound

s.W


thisprodu

ct.W

earapprop


entand

clothinginclud


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



contactw

ithopenwound

s.W


thisprodu

ct.W

earapprop


entand

clothinginclud


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



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

.

Poly(A) Tail-Length Assay Kit - Thermo Fisher Scientific · PDF fileAll reagents have been...

Documents

Transcript of Poly(A) Tail-Length Assay Kit - Thermo Fisher Scientific · PDF fileAll reagents have been...