nprot.2006.479

Amplification of 5¢ end cDNA with ‘new RACE’ Elizabeth Scotto-Lavino1,2, Guangwei Du2 & Michael A Frohman1,2 1Graduate Program in Molecular & Cellular Pharmacology, and 2Department of Pharmacological Sciences & Center for Developmental Genetics, Stony Brook University, Stony Brook, New York 11794, USA. Correspondence should be addressed to M.A.F. (michael@pharm.stonybrook.edu). Published online 25 January 2007; doi:10.1038/nprot.2006.479 ‘New RACE’ (rapid amplification of cDNA ends) PCR is a method for obtaining full-length cDNA for mRNA for which only part of the sequence is known. Starting with cellular mRNA, PCR is used to amplify regions between the known parts of the sequence and nonspecific tags at the ends of the cDNA. In ‘new RACE’, an anchor is ligated to the 5¢ end of the mRNA before reverse transcription, resulting in the selective production of full-length 5¢ cDNA ends. Although ‘new RACE’ can also be used to amplify 3¢ ends, only the protocol for obtaining 5¢ ends is presented here. This protocol can be completed in 1–3 days. INTRODUCTION The 5¢ end of mRNA is often difficult to obtain from conventional cDNA libraries. Obtaining a full-length sequence is important, however, for identifying potential cis-acting regulatory elements and for determining if there is more than one site of transcription initiation. The 5¢ untranslated sequences can also regulate transla- tional efficiency. ‘New RACE’, a variation of RNA ligase–mediated RACE1,2, departs from classical RACE3 in that the ‘anchor’ primer is attached to the 5¢ end of the mRNA before the reverse-transcription step; hence, the anchor sequence becomes incorporated into the first- strand cDNA if and only if the reverse transcription proceeds through the entire length of the mRNA of interest (and through the relatively short anchor sequence; see Fig. 3 in ref. 3). Before ‘new RACE’ is begun (Fig. 1), the mRNA is subjected to a dephos- phorylation step with shrimp alkaline phosphatase (SAP). This step does not affect full-length mRNAs, which have methylated ‘G’ caps at their termini, but it does dephosphorylate degraded mRNAs, which are uncapped at their termini4. Thus, degraded RNA is excluded from the ligation step later in the protocol. After the dephosphorylation step, the full-length mRNA is treated with tobacco acid pyrophosphatase (TAP). This removes the cap struc- ture but preserves the active phosphorylated 5¢ termini2,5. Using T4 RNA ligase, the full-length mRNA is then ligated to a short synthetic RNA oligonucleotide that has been generated by in vitro transcription of a linearized plasmid6. The RNA oligonucleotide– mRNA hybrids are then reverse-transcribed with a gene-specific primer, thus creating the first strand of cDNA. Finally, the 5¢ cDNA ends are amplified in two nested PCR reactions with additional gene-specific primers and primers derived from the sequence of the RNA oligonucleotide. ‘New RACE’ can also be used to generate 3¢ cDNA ends4,5 and is particularly useful for non-polyadenylated RNA. For this, cytoplas- mic RNA is dephosphorylated and is ligated to a short synthetic RNA oligonucleotide as described above. Although ligation of the oligonucleotide to the 5¢ end of the RNA was emphasized above, RNA oligonucleotides actually ligate to both ends of cytoplasmic RNA. For the reverse-transcription step, a primer derived from the RNA oligonucleotide sequence is used (e.g., the reverse comple- ment of NRC3, Fig. 1). Reverse transcription of the RNA oligo- nucleotides that are ligated to the 3¢ end of the cytoplasmic RNA results in the creation of cDNA with the RNA oligonucleotide sequence appended to the 3¢ end. Gene-specific primers oriented in the 5¢-3¢ direction and ‘new RACE’ primers (e.g., the reverse complements of NRC2 and NRC-1, Fig. 1), are used in nested PCR reactions to amplify the 3¢ ends. Here we provide a detailed protocol for the amplification of 5¢ ends by ‘new RACE’, including generation of the required reagents and the reverse-transcription and PCR steps. p u or G g n ih si lb uP er u ta N 700 2 © n at ur ep ro to co ls / m oc . er ut a n . w w w//:ptth NRC1,NRC2 1st- strand cDNA NRC1 NRC2 NRC1 NRC1 NRC2 NRC3 Polylinker T3 RN A po lym era se pro mo ter NRC2 RNA oligo NRC3 cDNA 5′ end 5′ 5′ 3′ 3′ Sma IGbx1 3′ UTR Sst I Pst I SmaI Gbx1 3′ UTR In vitro transcription GSP-RT GSP-RT GSP1 GSP2 * mRNA mRNA mRNA Dephosphorylation of degraded mRNA Removal of 5′ cap Ligation of RNA anchor oligo to 5′ end of mRNA First set of amplifications Reverse transcription Second set of amplifications a b c Figure 1 | The ‘new RACE‘ procedure. (a) Amplification of 5¢ partial cDNA ends. GSP, gene-specific primer; RT, reverse transcription. (b,c) In vitro synthesis of the RNA oligonucleotide used for ligation in ‘new RACE’ (b) and the corresponding required primers used (c). A 132-nt RNA oligonucleotide is produced by in vitro transcription of the plasmid shown using T3 RNA polymerase. Primers NRC1, NRC2 and NRC3 (sequences underlined) are derived from the sequence of the oligonucleotide. UTR, untranslated region. 3056 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS PROTOCOL MATERIALS REAGENTS .RNA sample (TAP-treated and untreated; see REAGENT SETUP) m CRITICAL All reagents must be RNase free. .Phosphatase buffer (10�) .DTT (0.1 M) .RNasin (40 U ml–1) .SAP (1 U ml–1; Fermentas) .Agarose gel (1%) in TAE buffer .Ethidium bromide .TAP buffer (10�) .TAP (5 U ml–1; Epicentre) .TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA, pH 8.0) .Phenol-chloroform (1:1 (vol/vol)) .Chloroform .Sodium acetate (3 M; pH 5.2) .Ethanol .Plasmid template DNA for transcribing RNA oligonucleotide .Restriction enzymes and buffers .Proteinase K .H20 treated with diethylpyrocarbonate (DEPC) .Transcription buffer (5�; as supplied by the manufacturer) .rUTP solution (10 mM) .rATP solution (10 mM) .rCTP solution (10 mM) .rGTP solution (10 mM) .DNA-dependent RNA polymerase (20 U ml–1) .Pancreatic DNase I (RNase-free) .Ligation buffer (10�: 500 mM Tris, pH 7.9, 100 mM MgCl2, 20 mM DTT and 1 mg ml–1 BSA) .RNA oligonucleotide (see REAGENT SETUP) .ATP (2 mM) .T4 RNA ligase (can be purchased from New England BioLabs; see REAGENT SETUP) .Reverse-transcription buffer (5�) .dNTP solution (containing all four dNTPs, each at 10 mM) .Gene-specific antisense primer (20 ng ml–1) .SuperScript II reverse transcriptase (Invitrogen) .RNase H .Hercules Hot-Start polymerase buffer (10�; Stratagene) m CRITICAL If the buffer already contains dNTPs, do not add additional nucleotides to the mixture. .Hercules Hot-Start polymerase (Stratagene) m CRITICAL It is necessary to use a hot-start protocol. .User-defined gene-specific oligonucleotide primers GSP1, GSP2, NRC1 and NRC2 (see REAGENT SETUP for primer design considerations and Figure 1 for details of primers NRC1 and NRC2) EQUIPMENT .Water baths or heating blocks preset to 37, 42, 50, 65 and 70 1C .Microcon spin filters (Millipore) REAGENT SETUP RNA sample The procedure described here uses relatively large amounts of RNA and can be ‘scaled down’ if RNA quantities are limited. If large quantities of RNA are used, it is practical to sacrifice an aliquot at each stage of the experiment to check for sample degradation. This can be achieved by agarose gel electro- phoresis. Samples with no detectable degradation can be stored indefinitely for future experiments. T4RNA ligase The 10� T4 RNA ligase buffers supplied by some manufacturers contain too much ATP6. Check the composition of any commercially supplied 10� buffer and if it contains more than 1 mM ATP, make your own using the same components, except adjust the ATP concentration to 1 mM (final 1� concentration should be 0.1 mM), or use the 10� buffer described in ref. 6. Synthesis of RNAoligonucleotide Choose a plasmid that can be linearized at a site about 100 bp downstream from a T7 or T3 RNA polymerase site (Fig. 1). Ideally, use a plasmid containing some insert cloned into the first polylinker site, because primers made from palindromic polylinker DNA do not do well in PCR. In the example presented here, pBS-SK-GBX-1-3¢UTR contains the 3¢ untranslated region of the mouse Gbx1 gene7 cloned into the SstI site of plasmid pBS-SK (Stratagene). This can be linearized with SmaI and transcribed with T3 RNA polymerase to produce a 132-nt RNA oligonucleotide (Steps 9–17 below). All but 17 of the nucleotides are from Gbx1. Adenosine residues are the best ‘acceptors’ for ligation of the 3¢ end of the RNA oligonucleotide to the 5¢ end of its target, if an appropriate restriction site can be found. Do a test transcription to ensure everything is working, then ‘scale up’. The oligonucleotide can be stored indefinitely at –80 1C for future experiments, and it is important to synthesize sufficient oligonucleotide to allow for losses due to purification and ‘spot-checks’ along the way. Alternatively, the RNA oligonucleotide can be ordered from a commercial source. PCR primer design for ‘new RACE’ The primers used for amplification are all derived from the RNA oligonucleotide (discussed above; Fig. 1). Primers should be selected with appropriate software, such as Primer3 from the Massachusetts Institute of Technology (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_ www.cgi), so that they have similar melting temperatures. PROCEDURE Dephosphorylation of degraded RNA � TIMING 2.5 h 1| In a sterile microfuge tube, combine the following reagents: m CRITICAL STEP In general, follow the manufacturer’s recommendations for use of the phosphatase. 2| Incubate the reaction for 1 h at 37 1C to dephosphorylate uncapped mRNA. This prevents degraded RNA fragments from participating in the subsequent ligation step. 3| Incubate the reaction for 15 min at 65 1C to inactivate SAP. Spin briefly in a microcentrifuge. ’ PAUSE POINT The products can be stored at –80 1C. 4| Analyze 2 mg (2 ml) of RNA by electrophoresis through a 1% agarose gel (TAE buffer) adjacent to a lane containing 2 mg of the original RNA preparation; stain the gel with ethidium bromide and visually confirm that the RNA remained intact during the dephosphorylation step. Degraded RNA will be present as a low-molecular-weight smear on the gel rather than a discrete band of high molecular weight. p u or G g n ih si lb uP er u ta N 700 2 © n at ur ep ro to co ls / m oc . er ut a n . w w w//:ptth Component Amount Final RNA 50 mg 10 mg 10� phosphatase buffer 5 ml 1� DTT (100 mM) 0.5 ml 50 mM RNasin (40 U ml–1) 1.25 ml 50 U SAP (1 U ml–1) 3.5 ml 3.5 U H20 to 50 ml – NATURE PROTOCOLS | VOL.1 NO.6 | 2006 | 3057 PROTOCOL Decapping of intact RNA � TIMING 3 h 5| In a sterile microfuge tube, mix the following reagents: m CRITICAL STEP Most protocols call for much more TAP. This enzyme is very expensive and it is not necessary to use more than the amount recommended in this protocol. 6| Incubate the reaction for 1 h at 37 1C, then add 200 ml TE buffer to stop the reaction. 7| Extract the reaction with a phenol-chloroform mixture, extract again with chloroform, and precipitate the RNA with 0.1 volume of 3 M sodium acetate, pH 5.2, and 2.5 volumes of ethanol. Resuspend the RNA in 40 ml H2O. Spin columns can be used as an alternative to phenol-chloroform extraction and ethanol precipitation. ’ PAUSE POINT The products can be stored at –80 1C. 8| Analyze 2 mg of RNA by electrophoresis through a 1% agarose gel (TAE buffer) adjacent to a lane containing 2 mg of the original RNA preparation; stain the gel with ethidium bromide and visually confirm that the RNA remained intact during the decapping step. RNA will be present as a low-molecular-weight smear on the gel rather than a discrete band of high molecular weight. Preparation of RNA oligonucleotide � TIMING 5 h 9| Linearize 25 mg of the plasmid to be transcribed (the plasmid should be reasonably free of RNase) by digestion with the appropriate restriction enzymes and buffers. 10| Treat the digestion reaction for 30 min at 37 1C with 50 mg ml–1 proteinase K to eliminate any residual RNase, extract twice with phenol-chloroform and once with chloroform, and collect the DNA by standard ethanol precipitation as described in Step 7. 11| Redissolve the template DNA in 25 ml TE buffer, pH 8.0. This will give a final concentration of approximately 1 mg ml–1. 12| In a sterile microfuge tube, at room temperature (25 1C), mix the transcription reagents in the order listed in Table 1. 13| Incubate for 1 h at 37 1C to allow transcription to occur. 14| After transcription, remove the DNA template by adding 0.5 ml DNase (RNase-free) for every 20 ml of reaction volume. Incubate for 10 min at 37 1C. 15| Check the oligonucleotide product by analyzing 5 ml of the test or preparative reaction by electrophoresis through a 1% agarose gel (TAE buffer). Expect to see a diffuse band of about the right size for the expected product (or a bit smaller) in addition to some smearing up and down the gel. 16| Purify the oligonucleotide by extraction with phenol-chloroform and then chloroform. Rinse three times with 1 ml H2O and then pass though a Microcon spin filter (prerinsed with H2O). p u or G g n ih si lb uP er u ta N 700 2 © n at ur ep ro to co ls / m oc . er ut a n . w w w//:ptth TABLE 1 | Transcription procedure. Component Amount (test scale) Final Amount (preparative scale) Final DEPC-treated H2O 4 ml – 80 ml – Transcription buffer (5�) 2 ml 1� 40 ml 1� DTT (0.1 M) 1 ml 100 mM 20 ml 100 mM rUTP (10 mM) 0.5 ml 0.5 mM 10 ml 0.5 mM rATP (10 mM) 0.5 ml 0.5 mM 10 ml 0.5 mM rCTP (10 mM) 0.5 ml 0.5 mM 10 ml 0.5 mM rGTP (10 mM) 0.5 ml 0.5 mM 10 ml 0.5 mM Linearized DNA from Step 11 (1 mg ml–1) 0.5 ml 0.5 mg 10 ml 10 mg RNasin (40 U ul–1) 0.25 ml 10 U 5 ml 200 U RNA polymerase (20 U ml–1) 0.25 ml 5 U 5 ml 100 U Total 10 ml – 200 ml – Component Amount Final RNA (obtained in Step 4 above) 42 mg 42 mg TAP buffer (10�) 5 ml 1� DTT (0.1 M) 0.5 ml 50 mM RNasin (40 U ml–1) 1.25 ml 50 U TAP (5 U ml–1) 1 ml 5 U H20 to 50 ml – 3058 | VOL.1 NO.6 | 2006 | NATURE PROTOCOLS PROTOCOL m CRITICAL STEP Microcon 10 spin filters have a cutoff size of 20 nt, and Microcon 30 spin filters have a cutoff size of 60 nt. Microcon 10 spin filters are most appropriate if the oligonucleotide is smaller than 100 nt, and Microcon 30 spin filters are most appropriate for anything larger. 17| Analyze a second aliquot of the oligonucleotide by electrophoresis through a 1% agarose gel (TAE buffer) to check the integrity and concentration of the sample. The oligonucleotide distribution pattern should look like that in Step 15 above; if it is much smaller, this indicates that degradation has occurred and the procedure should be repeated with fresh material. ’ PAUSE POINT Store in aliquots at –80 1C indefinitely. RNA oligonucleotide–cellular RNA ligation � TIMING 1 d 18| Set up two sterile microfuge tubes as follows: one with TAP-treated cellular RNA; the other with untreated cellular RNA (negative control). 19| Incubate for 16 h or overnight at 17 1C. 20| Purify the ligation product by spin filtration with a Microcon 100 spin filter (three times in H2O; prerinse filter with RNase-free H2O). The volume recovered should not exceed 20 ml. ’ PAUSE POINT The products can be stored at –80 1C. 21| Check integrity of the ligated RNA by analyzing 1/3 of the product by electrophoresis through a 1% agarose gel (TAE buffer). It should again look like the previous samples, as discussed in Step 15 above. Reverse transcription � TIMING 2 h 22| In a sterile microfuge tube, assemble the following transcription components on ice for each experimental and control sample from Step 20: 23| In a separate tube, add 20 ng of a gene-specific antisense primer (GSP-RT, Fig. 1) to the remaining RNA (about 6.7 mg) from Step 20 in 13 ml H20. Incubate for 3 min at 80 1C, cool rapidly on ice and centrifuge for 5 s. If a control reaction is desired, set up an identical tube in parallel to which reverse transcriptase is not added in Step 24. 24| Add the RNA-primer mix to the reverse-transcription components, then add 1 ml (200 U) of SuperScript II reverse transcriptase. Incubate for 1 h at 42 1C and for 10 min at 50 1C. 25| Inactivate the reverse transcriptase by incubating for 15 min at 70 1C. Centrifuge for 5 s. 26| Destroy the RNA template by adding 0.75 ml (1.5 U) of RNase H. Incubate for 20 min at 37 1C. 27| Dilute the reaction mixture to 100 ml with TE buffer and store at 4 1C (this is the 5¢-end oligonucleotide–cDNA pool). ’ PAUSE POINT The 5¢-end oligonucleotide–cDNA pool can be stored indefinitely at 4 1C. Avoid storing at –20 1C, which could snap some of the cDNA strands. First-round amplification � TIMING 2.5 h 28| In a sterile, 0.2-ml microfuge tube, mix the following reagents for each experimental and control sample from Step 27: p u or G g n ih si lb uP er u ta N 700 2 © n at ur ep ro to co ls / m oc . er ut a n . w w w//:ptth Component Amount Final Ligation buffer (10�) 3 ml 1� RNasin (40 U/ml) 0.75 ml 30 U RNA oligonucleotidea 4 mg 4 mg TAP-treated or untreated RNA 10 mg 10 mg ATP (2 mM) 1.5 ml 0.1 mM T4 RNA ligase (20 U ml–1) 1.5 ml 30 U H20 to 30 ml – aRNA oligonucleotides are at a molar excess of 3–6 over target cellular RNA. Component Amount Final Reverse-transcription buffer (5�) 4 ml 1� dNTP mixture (containing all four dNTPs, each at 10 mM) 1 ml 1.37 mM DTT (0.1 M) 2 ml 13.8 mM RNasin (40 U ml–1) 0.25 ml 10 U Total 7.25 ml – NATURE PROTOCOLS | VOL.1 NO.6 | 2006 | 3059 PROTOCOL 29| Add a 1-ml aliquot of the 5¢-end oligonucleotide–cDNA pool and 25 pmol each of primers GSP1 and NRC1. Set up a ‘no-template’ control as well. 30| Heat in a DNA thermal cycler for 5 min at 98 1C to denature the first-strand products and to activate the polymerase. Cool for 2 min to the appropriate annealing temperature (56–68 1C). Extend the cDNA for 40 min at 72 1C. 31| Carry out 35 cycles of amplification with a ‘step’ program as follows: ’ PAUSE POINT The products can be stored at 4 1C. Second-round amplification � TIMING 2.5 h. The entire procedure can be completed in 3 d but will take up to 5 d if the procedure is stopped according to the’ PAUSE POINT instructions. 32| Dilute a portion of the amplification products from the first round 1:20 in TE buffer. m CRITICAL STEP A second round of amplification is required because the use of only one gene-specific primer (in combination with a universal primer that binds to all of the cDNA templates present in the starting mixture) results in a substantial yield of nonspecifically amplified products. The second round, which uses a second gene-specific primer (again in combination with a universal primer), eliminates most or all of the nonspecific products. 33| In a sterile, 0.2-ml microfuge tube, mix the following reagents on ice: 34| Add a 1-ml aliquot of the diluted first-round amplification products (obtained in Step 32 above) and 25 pmol each of primers GSP2 and NRC2 (Fig. 1). Set up a ‘no-template’ control as well. 35| Mix and heat in a DNA thermal cycler for 5 min at 98 1C to denature the first-strand products and to activate the polymerase. 36| Carry out 30 cycles of amplification with a ‘step’ program as follows: ’ PAUSE POINT The products can be stored at 4 1C. 37| Separate 20% of the products of first- and second-round amplification by electrophoresis through a 1% agarose gel and check for specific partial cDNAs by Southern blot analysis; hybridize with a labeled oligomer or gene fragment derived from the amplification template (e.g., GSP-Hyb/Seq in Fig. 1a,b). Use the information gained from this analysis to optimize the RACE procedure (see ANTICIPATED RESULTS). ? TROUBLESHOOTING Problems with nonspecific amplification products Optimize the annealing temperature by gradually increasing it (about 2 1C at a time) at each stage of the proce