3' End cDNA amplification using classic RACE

3¢End cDNA amplification using classic RACEElizabeth Scotto–Lavino 1,2,Guangwei Du 2&Michael A Frohman 1,2,3GraduateProgram in Molecular &Cellular Pharmacology;Department of Pharmacological Sciences &Center for Developmental Genetics,Stony Brook University,Stony Brook,New York 11794,U.S.A.3Correspondence should be addressed to M.A.F.(michael@).Published online 11January 2007;doi:10.1038/nprot.2006.481Having knowledge of the entire 3¢sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA.Also,some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties,or change the encoded protein.Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE)PCR as long as part of the mRNA sequence is known;adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified.In 3¢RACE,the poly(A)tail functions as a non-specific tag at the 3¢end of the mRNA.cDNA ends can be obtained in 1–3days using this protocol.INTRODUCTIONPCR can be used to obtain cDNA fragments even when the sequence is known for only one end of the region to be amplified.This can be done by taking advantage of a natural or synthetic anchor sequence appended to the unknown end of the region 1.This approach can be used for either the 3¢end as described here,or the 5¢end as described in accompanying protocols 2,3.The 3¢ends of cDNA often contain signals that regulate the stability or subcellular localization of mRNAs,and sometimes alternative genomic sites are used for cleavage and polyadenylation,which can alter these aspects or change the encoded protein.3¢end sequences can be missing in several circumstances.First,some organisms do not employ polyadenylation to generate mRNAs;therefore standard poly(A)-tail-constructed libraries do not exist (for amplification by RACE,an anchor has to be added using RNA ligase).Second,cDNAs are sometimes identified in non-sequenced species using homology approaches to amplify regions that correspond to conserved protein stretches;in this case both the 3¢and 5¢ends of the cDNAs are missing.Third,alternative splicing,RNase-H sensitive sites,or A-rich truncation events can result in incorrect identification of the genuine 3¢end for cDNAs,even for sequenced and well-characterized genomes.3¢RACE can be used to quickly obtain missing 3¢end sequences.T o generate 3¢ends,mRNA is reverse transcribed using a primer (Q total ,Q T )that terminates in two mixed bases (GATC /GAC)followed by 17Ts and a unique primer sequence (Q I -Q O ;Fig.1a,b ).The end is amplified using a primer that contains part of this sequence (Q outer ,Q O )and that binds to these cDNAs at their 3¢ends,and a primer that matches the gene of interest (gene-specific primer 1;GSP1).A second amplification series is then performed using internal primers (Q inner (Q I )and GSP2)to suppress the amplification of non-specific products.The protocol described in this article provides a classic and simple means to generate 3¢end cDNA fragments,from reverse transcription through to amplification,and takes less than a day to perform.Experimental design considerations for 3¢RACEAn important factor in the generation of full-length 3¢-end partial cDNAs is the stringency of the reverse transcription reaction.Historically,reverse transcription reactions were carried out at relatively low temperatures (37–421C),using an excess of primer (B 1/2the mass of the mRNA,which represents an B 30:1molar ratio).Under these low stringency conditions,a stretch of A residues as short as 6–8nucleotides will suffice as a binding site for an oligonucleotide(dT)-tailed primer.This binding can result in the initiation of cDNA synthesis at sites upstream of the poly(A)tail,leading to truncation of the desired amplification product.One should be suspicious that this has occurred if a canonical poly-adenylation signal sequence is not found near the 3¢end of the cDNAs generated.The problem can be minimized by controlling two parameters:primer concentration and reaction temperature 4.The primer concentration can be reduced substantially withoutp u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s/m o c .e r u t a n .w w w //:p t t h mRNAabReverse transcriptionQ I Q O Q T Q I Q OQ TQ O Q I1st strand cDNAFirst set of amplificationsSecond set of amplifications"cDNA 3′ End"GSP1GSP2Q IQ O Q T5′Xho I Sst I Hind III3′*Figure 1|A schematic representation of 3¢RACE.See text for details.(a )Amplification of 3¢partial cDNA ends.(b )Schematic representation of the primers used in classic RACE.The 52nucleotide QT primer (5¢QO–QI–TTTT 3¢)contains a 17nucleotide oligo-(dT)sequence at the 3¢end followed by a 35nucleotide sequence that encodes Hind III,Sst I and Xho I recognition sites.The QI and QO primers overlap by 1nucleotide;the QI primer contains all three of the restriction enzyme recognition sites.Two additional nucleotides can be added to the 3¢end of QT to force it to bind to the junction of the cDNA and the poly(A)tail (G,A or C,followed by G,A,T or C).Primers:QT:5¢–ccagtgagcagagtgacgaggactcgagctcaagcttttttttttttttttt–3¢.QO:5¢–ccagtgagcagagtgacg–3¢.QI:5¢–gaggactcgagctcaagc–3¢.GSP1,gene-specific primer 1;GSP2,gene-specific primer 2;*-,GSP-Hyb/Seq (a gene-specific primer for use in hybridization and sequencing reactions).Figure modified with permission from ref.6.2742|VOL.1NO.6|2006|NATURE PROTOCOLSsignificantly decreasing the yield of cDNA 5.At lower concentra-tions,the primer will bind preferentially to the longest A-rich stretches present (such as the poly(A)tail).The quantity recom-mended in the protocol below represents a good starting point and can be reduced fivefold further if significant truncation is observed.In the protocol described below,the incubation temperature is raised slowly to encourage reverse transcription to proceed through regions of difficult secondary structure.Since the half-life of reverse transcriptase rapidly decreases as temperature increases,the reac-tion cannot be carried out at increased temperatures in its entirety.In theory,the problem of difficult secondary structure (and non-specific reverse transcription)could be ameliorated by using heat-stable reverse transcriptases,which are available from several suppliers.However,the authors have never been able to attribute improvements to the use of these enzymes,which are merely promiscuous DNA polymerases.Use of ‘Hot Start’PCR (through any of several approaches)is vital to minimize non-specific amplification.An annealing temperature close to the effective melting temperature of the primers should be used.The Q I and Q O primers work well at 641C under the PCR conditions recommended here,although these were optimized using legacy machines.Conditions may vary depending on the PCR machine used.GSPs of similar length and GC content should be puter programs to assist in the selection of primers are widely available and should be used —for example,Primer3(/cgi-bin/primer/primer3_www.cgi/).The default parameters suggested by the program work well in our experience.Extension should be carried out for 1minute for every 1,000bp of the expected product.If the expected product length is unknown,extension for 3–4minutes is a good starting point.Very little substrate is required for the PCR.1m g of poly(A)+RNA typically contains B 5Â107copies of each low-abundance transcript.The PCR described here works optimally when 103–105templates (of the desired cDNA)are present in the starting mixture;so,as little as 0.002%of the reverse transcription mixture suffices for the reaction.Addition of too much starting material will lead to the production of large amounts of non-specific product and should be avoided.The RACE technique is particularly sensitive to this problem,because every cDNA in the mixture,desired and undesired,contains a binding site for the Q O and Q I primers.It was empirically found that allowing extra extension time (40minutes)during the first amplification round (when the second strand of cDNA is created)can result in increased yields of the specific product relative to background amplification.In particu-lar,the extra extension time often leads to increased yields of long cDNAs versus short cDNAs when specific cDNA ends of multiple lengths were present 1.Prior treatment of cDNA templates with RNA hydrolysis,or a combination of RNAse H and RNAse A,occasionally improves the efficiency of specific cDNA amplification.MATERIALSm CRITICAL The materials required for this procedure can be purchased,along with the appropriate 5Âor 10Âenzyme reaction buffers,from most major suppliers.Enzymes are used as directed by the suppliers.REAGENTS.dNTP solution (containing all four dNTPs,each at 10mM).DTT (0.1M).Tris–EDTA solution (10mM Tris-HCl [pH 7.5],1mM EDTA [pH 8.0]).Reverse transcription buffer,5Â(as supplied by manufacturer).RNase H (Invitrogen).RNasin (Promega Biotech).SuperScript II reverse transcriptase (Invitrogen).Poly(A)+RNA,or total RNA.Poly(A)+RNA is used in preference to total RNA for reverse transcription to reduce background,but it is unnecessary to prepare it if only total RNA is available..Hercules Hot-Start polymerase (Stratagene)m CRITICAL It is necessary to use a Hot-Start protocol..Hercules Hot-Start polymerase buffer (10Â,Stratagene).m CRITICAL dNTPs can be purchased as 100mM solutions from many suppliers if the PCR buffer does not contain it.If the buffer contains dNTPs already,do not add additional nucleotides to the mixture.Note that any of many different heat-stable DNA polymerases can be used,although investigators should look for robust amplification (as opposed to fidelity or cost).Expand from Boehringer Mannheim is another good choice..Oligonucleotide primers,such as those described below (also see REAGENTSETUP ):EQUIPMENT.Water baths or heating blocks preset to 37,42,50,70and 801C .Programmable thermal cyclerREAGENT SETUPPrimer design for 3¢RACE User-specific primer sequences should be designed using on-line software to match the melting temperature (T m )of the RACE primers.A useful tool is Primer3,which was developed at MIT (/bioapps/primer3_www.cgi).Primers can be used‘crude’except for Q T ,which should be purified to ensure that it is uniformly full length (this can be requested as part of the commercial service from most companies that synthesize primers).PROCEDUREReverse transcription to generate cDNA templates1|Add the following transcription components to a sterile microcentrifuge tube on ice:p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s/m o c .e r u t a n .w w w //:p t t h Primer sequencePrimerconcentration Q T 5¢–CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT–3¢100ng m l –1Q O 5¢–CCAGTGAGCAGAGTGACG–3¢100ng m l –1Q I 5¢–GAGGACTCGAGCTCAAGC–3¢100ng m l –1GSP1and GSP2User-specific 25pmol m l –1ComponentAmount Final*Reverse transcription buffer,5x 4m l 1x dNTP solution (10mM)1m l 1.3mM DTT (0.1M)2m l 12.9mM Q T primer (100ng/m l)0.5m l 50ng RNasin (40U/m l)0.25m l 10U TOTAL7.75m l–*Final concentration refers to concentration following addition of components from Steps 2and 3.NATURE PROTOCOLS |VOL.1NO.6|2006|27432|In a separate microcentrifuge tube,add 1m g of poly(A)+RNA,or 5m g of total RNA,to make a final volume of 11.25m l in distilled H 2O.Incubate at 801C for 3minutes,cool rapidly on ice and centrifuge at 10,000g (or maximum speed of microcentri-fuge)at 41C for 5seconds.3|Add the RNA to the reverse transcription components.Following this,add 1m l (200U)of SuperScript II reverse transcriptase,mix and incubate suspension for 5minutes at room temperature (B 22–251C),followed by 1hour at 421C and 10minutes at 501C.m CRITICAL STEP The incubation at room temperature ensures that the oligo-dT primer region anneals to the poly(A)-tail and undergoes extension during the 421C incubation.Although optimal enzyme stability and activity occurs at 371C,in some cases additional or more efficient extension of the pool of cDNAs of interest can be obtained by raising the extension temperature to 501C to remove mRNA secondary structures,despite the limited enzyme stability under these conditions.4|Inactivate the reverse transcriptase by incubating at 701C for 15minutes.Following incubation,microcentrifuge the suspension at room temperature for 5seconds at top speed.5|Destroy RNA template by adding 0.75m l (1.5U)of RNAse H and incubating at 371C for 20minutes.6|Dilute the reaction mixture to 1ml with Tris–EDTA solution.This is the 3¢end cDNA pool.!CAUTION To avoid damaging the cDNA,do not store at –201C.’PAUSE POINT The 3¢end cDNA pool can be stored at 41C indefinitely.First-round amplification of reverse transcription endsTIMING 2h7|Add the following transcription components to a 0.2ml sterile microcentrifuge tube on ice:8|Add a 1m l aliquot of the 3¢end cDNA pool (obtained in Step 6above)and 25pmols each of primers GSP1and Q O .Include a control reaction containing all components except for the 3¢end cDNA pool,to confirm that the products generated in the experimental tube do not result from contaminations in the reagents.9|Mix and heat the suspension in a DNA thermal cycler for 5minutes at 981C to denature the first strand products and acti-vate the polymerase.Cool to the appropriate annealing temperature (B 56–681C,depending on the T m of GSP1)for 2minutes.Extend the cDNAs at 721C or 40minutes.m CRITICAL STEP The lengthy extension time is helpful because the first round of synthesis is inefficient,probably owing to RNA fragments bound to the cDNA.10|Carry out 30cycles of amplification as follows:’PAUSE POINT The reaction mixture is stable indefinitely at room temperature.Second round amplification11|Dilute a portion of the amplification products from the first round (Step 10)1:20in Tris–EDTA solution.m CRITICAL STEP A second round of amplification is required because the use of only one GSP (in combination with a universal primer that binds to all of the cDNA templates present in the starting mixture)results in a significant yield of non-specifically amplified products.The second round,which employs a second GSP (again in combination with a universal primer),eliminates most to all of the non-specific products.12|Mix the following transcription components in a 0.2ml sterile microcentrifuge tube on ice:p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s/m o c .e r u t a n .w w w //:p t t h ComponentAmountFinal Hercules Hot-Start polymerase buffer (10Â)5m l 1ÂdNTP solution (10mM)1.0m l 200m M Hercules Hot-Start polymerase2.5U 0.05U H 20to 50m l–Cycle number DenaturationAnnealingPolymerization/extension 1–2910seconds at 941C 10seconds at 52–681C 3minutes at 721C3010seconds at 941C10seconds at 52–681C15minutes at 721C,then cool to room temperatureComponentAmountFinal Hercules Hot-Start polymerase buffer (10x)5m l 1xdNTP solution (10mM)1.0m l 200m M Hercules Hot-Start polymerase2.5U 0.05U H 20to 50m l–2744|VOL.1NO.6|2006|NATURE PROTOCOLS13|Add a 1m l aliquot of the diluted first round amplification products from Step 11and 25pmols each of primers GSP2and Q I .Include a control reaction containing all components except for the 3¢end cDNA pool,to confirm that the products generated in the experimental tube do not result from contaminations in the reagents.14|Mix and heat in a DNA thermal cycler for 5minutes at 981C to denature the first-strand products and activate the polymerase.15|Carry out 30cycles of amplification using a step program as follows:’PAUSE POINT The reaction mixture is stable indefinitely at room temperature.16|Analyze the final PCR products using gel electrophoresis.cDNA identities can be confirmed by hybridization using the known portion of the cDNA that overlaps the amplification product,or by sequencing the PCR products.TIMING3¢ends ready for sequence analysis can be obtained in less than one day.Taking advantage of the indicated Pause Points will lengthen the procedure to a minimum of 3days.Steps 1–6:2hours Steps 7–10:2hours Steps 11–16:2hours?TROUBLESHOOTINGFor common RACE issues concerning reverse transcription and amplification,please see refs.2,3.ANTICIPATED RESULTSRACE-PCR should lead to the generation of products that are predominantly derived from the gene of interest and capable of being sequenced directly,using a yet-more internal GSP.It should be noted,however,that in the event of alternately spliced or initiated terminated messages,it will be necessary to clone (or gel purify and re-amplify)the individual fragments to perform the analysis steps.Moreover,even a product that appears to consist of a single band should not be sequenced from the non-specific end —small differences in the numbers of A residues added and incorporated into the final product will result in sequences that are out of register as soon as the gene of interest is encountered.ACKNOWLEDGMENTS The authors thank Yue Zhang and the publisher for permission to use and adapt material from ‘‘RACE all the way to the end’’(ref.6).This work was supported by awards NIHDDK 64166and NIHGM71520to M.A.F.,and NIHGM071475and a Scientist Development Grant from the American Heart Association to G.D.(0430096N).COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.Published online at Reprints and permissions information is available online at /reprintsandpermissions 1.Frohman,M.A.,Dush,M.K.&Martin,G.R.Rapid production of full-length cDNAs from rare transcripts by amplification using a single gene-specific oligonucleotide primer.Proc.Natl A 85,8998–9002(1988).2.Scotto–Lavino,E.,Du,G.&Frohman,M.A.5¢End cDNA amplification using classic RACE.Nat.Protocols (2006).DOI:10.1038/nprot.2006.4803.Scotto–Lavino,E.,Du,G.&Frohman,M.A.5¢End cDNA amplification using new RACE.Nat.Protocols (2007).DOI:10.1038/nprot.2006.4794.Wickens,M.&Stephenson,P.Role of the conserved AAUAAA sequence:fourAAUAAA point mutants prevent mRNA 3¢end formation.Science 226,1045–1050(1984).5.Coleclough,e of primer-restriction end adapters in cDNA cloning.Methods Enzymol.154,64–83(1987).6.Zhang,Y.In Generation of cDNA Libraries (ed.Ying,S.-Y.)13–24(Humana Press,Totowa,NJ,2003).p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s/m o c .e r u t a n .w w w //:p t t h Cycle number DenaturationAnnealingPolymerization/extension 1–2910seconds at 941C 10seconds at 52–681C 3minutes at 721C3010seconds at 941C10seconds at 52–681C15minutes at 721C,then cool to room temperatureNATURE PROTOCOLS |VOL.1NO.6|2006|2745。