Roche Transcriptor First Strand cDNA Synthesis Kit instruction(1).pdf

www.roche-applied-For life science research only.Not for use in diagnostic procedures.Transcriptor First Strand cDNA Synthesis Kity Version 6.0Content version:September 2010Cat.No.04 379 012 001Kit for 50 reactions including 10 control reactionsCat.No.04 896 866 001Kit for 100 reactionsCat.No.04 897 030 001Kit for 200 reactionsStore the kit at?15 to?25CNStore Control RNA(vial 7 in Cat.No 04 379 012 001)at 70C or below.2www.roche-applied-04 379 012 001 Transcriptor First Strand cDNA Synthesis Kity Version 6.0Table of Contents1.What this Product Does.3Number of Tests 3Kit Contents 3Storage and Stability 4Additional Equipment and Reagents Required 4Application 42.How to Use this Product.52.1Before You Begin 5Precautions 5Sample Material 5Primer 62.2Procedure 8Standard RT-PCR Procedure 8Procedure A:cDNA Synthesis with anchored-oligo(dT)18 OR random OR sequence-specific primer 8Procedure B:cDNA Synthesis with anchored-oligo(dT)18 primer AND random hexamer primer 112.3Control Reaction 14cDNA Synthesis 14PCR for PBGD 15PCR in a conventional thermal block cycler 15PCR on the LightCycler Carousel-Based System 16PCR on the LightCycler 480 Instrument 183.Results.204.Troubleshooting.235.Additional Information on this Product.26How this Product Works 26References 27Quality Control 286.Supplementary Information.296.1Conventions 29Text Conventions 29Symbols 296.2Changes to Previous Version 296.3Ordering Information 296.4Trademarks 31PROTOCOLPROTOCOLwww.roche-applied-304 379 012 001 Transcriptor First Strand cDNA Synthesis Kit y Version 6.01.What this Product DoesNumber of TestsThe kit is designed for 50,100 or 200 reactions(depending on pack size).Kit ContentsVial/Cap LabelContenta)Cat.No.04 379 012 001 b)Cat.No.04 896 866 001 c)Cat.No.04 897 030 0011red Transcriptor Reverse Transcriptase a)1 vial,25?l(20 U/?l)b)1 vial,50?l (20 U/?l)c)2 vials,each 50?l(20 U/?l)Storage buffer:200 mM potassium phosphate,2 mM dithiothreitol,0.2%Triton X-100(v/v),50%glycerol(v/v),pH approx.7.2 2color-lessTranscriptor RT Reaction Buf-fer(5?)a)1 vial,1 ml b)1 vial,1 mlc)2 vials,each 1 ml 5?conc.:250 mM Tris/HCl,150 mM KCl,40 mM MgCl2,pH approx.8.5(25C)3color-less Protector RNase Inhibitor a)1 vial,50?l(40 U/?l)b)1 vial,100?l(40 U/?l)c)2 vials,each 100?l(40 U/?l)Storage buffer:20 mM Hepes-KOH,50 mM KCl,8 mM dithiothreitol,50%glycerol(v/v),pH approx.7.6(at 4C)4yellow/purple Deoxynuc-leo-tide Mix a)1 vial,100?l(yellow cap)b)1 vial,200?l(purple cap)c)2 vials,each 200?l(purple cap)10 mM each dATP,dCTP,dGTP,dTTP5blueAnchored-oligo(dT)18 Primera)1 vial,100?l(50?M)b)1 vial,200?l(50?M)c)2 vials,each 200?l(50?M)6blue Random Hexamer Primera)1 vial,100?l(600?M)b)1 vial,200?l (600?M)c)2 vials,each 200?l(600?M)7green Control RNA a)1 vial,20?l(50 ng/?l)contains a stabilized solution of a total RNA fraction purified from an immortalized cell line(K562)8greenControl Primer Mix PBGDa)1 vial,40?l 5?M forward and reverse primer specific for human porphobilinogen deaminase(PBGD)4www.roche-applied-04 379 012 001 Transcriptor First Strand cDNA Synthesis Kity Version 6.0Storage and StabilityStore the kit at-15 to-25C through the expiration date printed on the label.LThe Control RNA(vial 7 in Cat.No.04 379 012 001)should be stored at?70C.NAvoid repeated freezing and thawing.Additional Equipment and Reagents Required standard laboratory equipment nuclease-free,aerosol-resistant pipette tips pipettes with disposable,positive-displacement tips sterile reaction tubes for preparing master mixes and dilutions standard benchtop microcentrifuge thermal block cycler with a heated lid sequence-specific PCR primers(optional)for control reactions in combination with a LightCycler Instrument:LightCycler 1.5 Instrument*,LightCycler 2.0 Instrument*or LightCycler480 Instrument*available from Roche Applied ScienceApplicationThe Transcriptor First Strand cDNA Synthesis Kit is designed to reverse tran-scribe RNA(mRNA,total RNA,viral RNA,and in vitro transcribed RNA)from a variety of sources for the following applications:Study gene expression levels,via two-step RT-PCR,using qualitative RT-PCR on conventional thermal cyclers or quantitative RT-PCR on the LightCyclerCarousel-Based System,the LightCycler 480 System,or other real-time PCR instruments.Generate cDNA libraries with large and full-length inserts.Clone genes of interest.The kit contains all components required for cDNA reactions for use with con-ventional thermal cyclers and real-time PCR instruments.In addition,the 50-reaction pack size includes 10 control reactions.9(7 for b,c)color-less Water,PCR-gradea)1 vial,1 mlb)2 vials,each 1mlc)3 vials,each 1 mlLIn Cat.No.04 896 866 001 and Cat.No.04 897 030 001 the control reagents(vial 7 and 8)are not included.Therefore,in these kits vial 7 is Water,PCR Grade.Vial/Cap LabelContenta)Cat.No.04 379 012 001 b)Cat.No.04 896 866 001 c)Cat.No.04 897 030 0011.What this Product Does,continuedwww.roche-applied-504 379 012 001 Transcriptor First Strand cDNA Synthesis Kit y Version 6.02.How to Use this Product2.1Before You BeginPrecautionsSpecial precautions should be taken when working with RNA:Always wear gloves when working with RNA.After putting on gloves,do not touch surfaces and equipment to avoid reintroduction of RNases to decon-taminated material.Designate a special area for RNA work only.Treat surfaces of benches and glassware with commercially available RNase inactivating agents.Clean benches with 100%ethanol.Use commercially available sterile and RNase-free disposable plasticware only.Purchase reagents that are free of RNases.Reserve separate reagents for RNA work only.All solutions should be made with DEPC-treated H2O.All required reagents should be kept on ice.Extract RNA as quickly as possible after obtaining samples.For best results,use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at 70C.Sample MaterialTemplate RNA:Isolated total RNA,mRNA,viral RNA or in vitro transcribed RNA.NHigh quality intact RNA,free of residual genomic DNA,RNase,and inhibi-tors is essential for good results.In particular,take the following precau-tions to avoid contaminating RNA with RNase at any step in the isolation process(starting with cell lysis):Use either RNase inhibitors such as Protector RNase Inhibitor or isolation conditions that inactivate RNases.If necessary,analyze different steps in the process(lysis,isolation)by gel electrophoresis(ethidium bromide staining)to ensure that the sample is still RNase-free.Remember that RNases can also be present on contaminated glassware.To prepare total RNA or mRNA,we recommend using Roche Applied Science reagents.For a selection of products which produces high quality intact RNA templates suitable for RT-PCR please refer to section 6.Supplementary Infor-mation,Ordering Information or to our Special Interest Site on manual nucleic acid isolation and purification at www.roche-applied- information on automated nucleic acid isolation using the MagNA Pure LC System or the MagNA Pure Compact System,visit .6www.roche-applied-04 379 012 001 Transcriptor First Strand cDNA Synthesis Kity Version 6.0PrimerDepending on the type of analysis,to which the cDNA is to be subjected,use one of three different priming methods described below:Random hexamer primer:In general,to reversely transcribe 5?g of total RNA with random hexamers a final primer concentration of 60?M is sufficient.Increasing the concentra-tion of hexamers to higher concentrations for the transcription of 5?g RNA may increase yield of small PCR products(500 bp),but may decrease the yield of longer PCR products and full-length transcripts.Note that random hexamer priming is the most non-specific priming method and specificity is only obtained by PCR primers in the following PCR reaction.Anchored-oligo(dT)18 primer:As anchored-oligo(dT)18 primers are specific to the small pool of poly(A)+RNA in the whole total RNA pool(1-2%),the amount of cDNA resulting from reverse transcription reactions with anchored-oligo(dT)18 primers is consid-erably lower than with random hexamers.Anchored-oligo(dT)18 priming is recommended when performing RT-PCR for new mRNA targets.Anchored-oligo(dT)18 produces an RT-PCR product more consistently than random hexamers or gene-specific primers.Sequence-specific primer:The use of gene-specific primers(recommended final concentration is 2?M)is the most specific priming method,but sometimes fails to prime cDNA even though the same primers work in PCR on DNA templates.If gene-specific priming fails in RT-PCR,repeat first-strand synthesis using anchored-oligo(dT)18 primers.Type of RT PrimerBindsAdvantages/CommentsAnchored-oligo(dT)18Very begin-ning of the poly(A)tail Prevents priming from internal sites of the poly(A)tail.Generates full-length cDNA.Preferred priming method for most two-step RT-PCR.Available as part of the Transcriptor First Strand cDNA Synthesis Kit only.Random hexamerMany sites throughout the length of an RNA Provides uniform representation of all RNA sequences in mRNA.Can prime cDNA transcription from RNAs that do not carry a poly(A)tail.The ratio of random primers to RNA in the RT reaction determines the average length of cDNAs generated.Example:A high ratio will generate relatively short cDNAs,increasing the chance of copying the complete target sequence.Short cDNA transcripts may help to over-come difficulties caused by RNA secondary structures.2.1Before You Begin,continuedwww.roche-applied-704 379 012 001 Transcriptor First Strand cDNA Synthesis Kit y Version 6.0Whenever possible,design primers that anneal to exon sequences on both sides of an intron or on exon/exon boundaries.This will allow differentiation of the amplified cDNA from contaminating genomic DNA because amplification of DNA will result in longer amplicons due to the additional intron sequence.NPrimers should not be self-complementary.Sequence-specificOnly sequences that are exactly com-plementary to the primer sequence Selects for a particular RNA.Greatly increases the specificity of the RT-PCR.Type of RT PrimerBindsAdvantages/Comments2.1Before You Begin,continued8www.roche-applied-04 379 012 001 Transcriptor First Strand cDNA Synthesis Kity Version 6.02.2ProcedureStandard RT-PCR ProcedureTwo different procedures are provided:A:Reverse transcription using either anchored-oligo(dT)18 priming OR ran-dom hexamer priming OR sequence-specific priming.In the majority of cases,cDNA is generated with only one sort of primers.B:Reverse transcription using a combination of anchored-oligo(dT)18 prim-ing AND random hexamer priming.This can be the method of choice to increase sensitivity,but the specificity of the reaction might be reduced com-pared to single anchored-oligo(dT)18 priming.LDepending on which type of primer system you have decided to use,fol-low Procedure A or B described below.If you are going to use a sequence-specific primer follow Procedure A.NPreheat the thermal block cycler to the temperature of the RT reaction(see step 6 below)or set-up the experimental protocol for the LightCyclerInstrument before starting the procedure.Procedure A:cDNA Synthesis with anchored-oligo(dT)18 primer OR random OR sequence-specific primerThe following conditions describe a first-strand cDNA synthesis for a two-step RT-PCR.Fig.1 shows the standard procedure for cDNA synthesis used for sin-gle reactions and the simplified one if multiple reactions should be performed.Fig.1:Overview of cDNA synthesis procedures in single and multiple reactionsStandard RT-PCR Reactionwww.roche-applied-904 379 012 001 Transcriptor First Strand cDNA Synthesis Kit y Version 6.0?Thaw all frozen reagents before use.Briefly centrifuge them before starting the procedure.Keep all reagents on ice while setting up the reactions.?In a sterile,nuclease-free,thin-walled PCR tube on ice,prepare the template-primer mixture for one 20?l reaction by adding the compo-nents in the order listed below.NAlways wear gloves when handling RNA.Template-Primer Mix(for 1 reaction)ComponentVol.Final conc.total RNA orpoly(A)+mRNA1?g total RNA or10 ng poly(A)+RNAa)Primer choose either:Anchored-oligo(dT)18 Primer,50 pmol/?l(vial 5)1?l2.5?MOR Random Hexamer Primer,600 pmol/?l(vial 6)2?l60?MOR Sequence-Specific Primervariable0.5-2.5?MWater,PCR-grade(vials 7 or 9)variableto make total volume=13?lTotal volume13?la)These are the suggested concentrations for initial experiments.Suitable template concentrations may range from 10 ng to 5 g total RNA and from 1 to 100 ng mRNA.LWhen working with low concentrated RNA samples(4 kb60 min at 50CRandom hexamer primers,600 pmol/?lUp to 4 kb10 min at 25C,followed by 30 min at 55C4 kb10 min at 25C,followed by 60 min at 50C?Inactivate Transcriptor Reverse Transcriptase by heating to 85 for 5 min.Stop the reaction by placing the tube on ice.At this point the reaction tube may be stored at+2 to+8C for 1-2 h or at-15 to-25C for longer periods.2.2Procedure,continuedStandard RT-PCR Reaction:Procedure Awww.roche-applied-1104 379 012 001 Transcriptor First Strand cDNA Synthesis Kit y Version 6.0NThe final MgCl2 concentration in the reverse transcription reaction is 8 mM.Therefore,each?l of the cDNA contributes 8 nmol MgCl2 to the fol-lowing reaction.Optimize the MgCl2 concentration of the PCR if necessary.NTranscriptor RTase has RNase H activity.RNase H removes the RNA tem-plate after cDNA synthesis,allowing PCR primers to more easily bind the cDNA,which in some cases increases the sensitivity of the PCR(Polumuri et al.,2002).Procedure B:cDNA Synthesis with anchored-oligo(dT)18 primer AND random hexamer primerThe following conditions describe a first strand cDNA synthesis for a two-step RT-PCR with a mixture of anchored-oligo(dT)18 primer AND random hexamer primers.?For PCR:The cDNA can be added to the PCR without purification.In general,use 1 5?l of the reaction product(first-strand cDNA)as a template for PCR.For initial experiments,try using 2?l cDNA template for a 50?l PCR.For PCR on one of the LightCycler instruments,use 2 5?l of the cDNA reaction or dilutions in a 20?l reaction.LThe cDNA product does not need to be purified before it is used in PCR.?Thaw all frozen reagents before use.Briefly centrifuge them before starting the procedure.Keep all reagents on ice while setting up the reactions.2.2Procedure,continuedStandard RT-PCR Reaction:Procedure B12www.roche-applied-04 379 012 001 Transcriptor First Strand cDNA Synthesis Kity Version 6.0?In a sterile,nuclease-free,thin-walled PCR tube on ice,prepare the template-primer mixture for one 20 l reaction by adding the compo-nents in the order listed below.NAlways wear gloves when handling RNA.Template-Primer Mix(for 1 reaction)ComponentVol.Final conc.total RNA orpoly(A)+mRNA1?g total RNA or10 ng poly(A)+RNAa)Anchored-oligo(dT)18 primer,50 pmol/?l(vial 5)1?l2.5?MAND random hexamer primer,600 pmol/?l(vial 6)2?l60?MWater,PCR-grade(vials 7 or 9)variableto make total volume=13?lTotal volume13?la)These are the suggested concentrations for initial experiments