3 RevertAid First Strand cdna Synthesis Kit (#K1621 for 10 reactions) Kit is designed for preparation of full-length fi rst strand cdna from RNA templates. The RevertAid fi rst strand cdna synthesis kit relies on a genetically engineered version of the Moloney Murine Leukemia Virus reverse transcriptase (RevertAid M-MuLV RT) with low RNase H activity. This allows the synthesis of full-length cdna from long templates (up to 13 kb). RevertAid M-MuLV RT synthesizes fi rst strand cdna at sites determined by the type of primer used: random hexamer primer: at non-specifi c points along an RNA template. In this case, all RNA s in a population are templates for cdna synthesis. : at the 3 -end of poly(a) + mrna. In this case, only mrna s with 3 -poly(a) tails are templates for cdna synthesis. sequence specifi c primer: at a primer-binding site. First strand cdna synthesized with this system can be used as a template in the polymerase chain reaction (PCR)*. As the reaction conditions of cdna synthesis and PCR are compatible, cdna reaction mixture can be added directly to a PCR mixture. The fi rst strand cdna synthesized can also be used as a template for second strand synthesis. Radiolabeled DNA can be used as a probe in hybridization experiments. 1.1 kb RNA with 3 -poly(a) tail is provided as a control. * The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche, Inc. 4 COMPONENTS OF THE KIT 1. RevertAid M-MuLV Reverse Transcriptase (200u*/µl): 15µl of the enzyme solution in storage buffer: 50mM Tris-HCl (ph 8.3), 0.1M NaCl, 1mM EDTA, 5mM DTT, 0.1% Triton X-100 and 50% glycerol. 2. RiboLock Ribonuclease Inhibitor (20u**/µl): 15µl of the enzyme solution in storage buffer: 20mM HEPES-NaOH (ph 7.5), 50mM NaCl, 8mM DTT, 0.5mM ELUGENT Detergent and 50% glycerol. 3. 5x Reaction Buffer: 100µl of 5x reaction buffer: 250mM Tris-HCl (ph 8.3 at 25 C), 250mM KCl, 20mM MgCl 2, 50mM DTT. 4. 10mM dntp Mix: 30µl of 10mM aqueous solution of each dgtp, datp, dttp, dctp. 5. Oligo(dT) 18 Primer: 15µl of 0.5µg/µl (15A 260 units/ml) aqueous solution. 6. Random Hexamer Primer: 15µl of 0.2µg/µl (6A 260 units/ml) aqueous solution. 7. Control Primer: 15µl of 10pmol/µl (1.7A 260 units/ml) 17-mer aqueous solution. 8. Control RNA: 15µl of 1.1 kb RNA with 3 -poly(a) tail, 0.5µg/µl. 9. DEPC-treated Water: 2 x 1.5ml of water deionized on a Milli-Q system and treated with DEPC. * One unit of RevertAid M-MuLV RT incorporates 1 nanomole of dtmp into a polynucleotide fraction (adsorbed on DE-81) in 10min at 37 C. ** One unit of RiboLock Ribonuclease Inhibitor inhibits the activity of 5ng RNase A by 50%.
5 PROCEDURES 1. Synthesis of First Strand cdna Suitable for PCR Amplification! Prepare the following reaction mixture in a tube on ice: template RNA*: total RNA 0.1-5µg or poly(a) + RNA 10ng-0.5µg or specifi c RNA 0.01pg - 0.5µg primer: primer (0.5µg/µl) 1µl or random hexamer primer (0.2µg/µl) 1µl or sequence-specifi c primer 15 20pmol DEPC-treated water to 12µl Mix gently and spin down for 3-5sec. in a microcentrifuge. " Incubate the mixture at 70 C for 5min, chill on ice and collect drops by brief centrifugation. # Place the tube on ice and add the following components in the indicated order: 5x reaction buffer 4µl RiboLock Ribonuclease Inhibitor (20u/µl) 1µl 10mM dntp mix 2µl Mix gently and collect drops by brief centrifugation. $ Incubate at 37 C for 5min (at 25 C for 5min if random hexamer primer is used). * The amount of total RNA or poly(a) + RNA required for reaction depends on the level of expression of a gene. 6 % Add RevertAid M-MuLV Reverse Transcriptase (200u/µl) 1µl Final volume 20µl & Incubate the mixture at 42 C for 60min (incubate at 25 C for 10min and fi nally at 42 C for 60min if the random hexamer primer is used). ' Stop the reaction by heating at 70 C for 10min. Chill on ice. The fi rst strand cdna synthesized can be used directly for amplifi cation by PCR. The PCR could be performed using the following products: Taq DNA Polymerase (recombinant) (#EP0401, #EP0402, #EP0403, #EP0404); Taq DNA Polymerase (native, without BSA) (#EP0281, #EP0282, #EP0283, #EP0284); Taq DNA Polymerase (native, with BSA) (#EP0071, #EP0072); 2mM dntp Mix (#R0241, #R0242); 10mM dntp Mix (#R0191, #R0192). Notes 1. Prior separation of poly(a) + RNA from total RNA is not mondatory, however, doing so may improve the yield and purity of the fi nal product. 2. RNA sample must be free of contaminating genomic DNA. 3. Unlike the priming, which usually requires no optimization, the ratio of a random hexamer primer to RNA is critical in terms of the average length of cdna synthesized in the reaction. Increasing the ratio of hexamer/rna will result in the higher yield of shorter (~500 bp) cdna, whereas decreasing this ratio will produce longer products. 4. Problems with secondary structure of GC rich mrna can be alleviated by increasing the reaction temperature up to 45 C. 5. For analysis of the reaction products (see chapter 4) cdna should be radiolabeled with [ 32 P]. For this purpose, add 10µCi
7 [α- 32 P]dNTP (e.g., datp) to the reaction mixture prior to adding RevertAid M-MuLV RT. Stop the reaction by adding 1µl of 0.5M EDTA and keep on ice. 6. The synthesized cdna should be stored at -20 C. 2. Synthesis of First Strand cdna Suitable for Second Strand Synthesis! Prepare the following reaction mixture in a tube on ice: template RNA: poly(a) + RNA 1µg or specifi c RNA 0.5-1µg primer: primer (0.5µg/µl) 1µl or random hexamer primer (0.2µg/µl) 1µl or sequence-specifi c primer 100pmol DEPC-treated water to 12µl Mix gently and spin down for 3-5sec. in a microcentrifuge. " Incubate the mixture at 70 C for 5min, chill on ice and collect drops by brief centrifugation. # Place the tube on ice and add the following components in the indicated order: 5x reaction buffer 4µl RiboLock Ribonuclease Inhibitor (20u/µl) 1µl 10mM dntp mix 2µl Mix gently and collect drops by brief centrifugation. $ Incubate at 37 C for 5min (at 25 C for 5min if random hexamer primer is used). % Add RevertAid M-MuLV Reverse Transcriptase (200u/µl) 1µl Final volume 20µl 8 & Incubate the mixture at 42 C for 60min (incubate at 25 C for 10min and fi nally at 42 C for 60min if the random hexamer primer is used). ' Stop the reaction by heating at 70 C for 10min. Chill on ice. The fi rst strand cdna synthesized can be used for the second strand synthesis, which could be performed with the following products: DNA Polymerase I (#EP0041, #EP0042); T4 DNA Ligase (#EL0014, #EL0011, #EL0012); Ribonuclease H (#EN0201, #EN0202); S1 Nuclease (#EN0321). Notes 1. To increase the specifi city and effi ciency of cdna synthesis it is necessary to isolate poly(a) + fraction from total cellular RNA. 2. Unlike the priming, which usually requires no optimization, the ratio of a random hexamer primer to RNA is critical in terms of the average length of cdna synthesized in the reaction. Increasing the ratio of hexamer/rna will result in the higher yield of shorter (~500 bp) cdna, whereas decreasing this ratio will produce longer products. 3. Problems with secondary structure of GC rich mrna can be alleviated by increasing the reaction temperature up to 45 C. 4. For analysis of the reaction products (see chapter 4) cdna should be radiolabeled with [ 32 P]. For this purpose, add 10µCi [α- 32 P]dNTP (e.g., datp) to the reaction mixture prior to adding RevertAid M-MuLV RT. Stop the reaction by adding 1µl of 0.5M EDTA and keep on ice. 5. The synthesized cdna should be stored at -20 C.
9 3. Synthesis of First Strand cdna Radiolabeled to High Specific Activity! Prepare the following reaction mixture in a tube on ice: template RNA: poly(a) + RNA 0.5µg primer: primer (0.5µg/µl) 1µl or random hexamer primer (0.2µg/µl) 1.5µl or sequence-specifi c primer 100pmol DEPC-treated water to 8µl Mix gently and spin down for 3-5sec. in a microcentrifuge. " Incubate the mixture at 70 C for 5min, chill on ice and collect drops by brief centrifugation. # Place the tube on ice and add the following components in the indicated order: 5x reaction buffer 4µl RiboLock Ribonuclease Inhibitor (20u/µl) 1µl 10mM dgtp, dctp, dttp mix* 1µl 0.1mM datp* 4µl Mix gently and collect drops by brief centrifugation. $ Incubate at 37 C for 5min (at 25 C for 5min if random hexamer primer is used). % Add: [α- 32 P]dATP (10mCi/ml) 1µl RevertAid M-MuLV Reverse Transcriptase (200u/µl) 1µl Final volume 20µl * Individual dntp solutions are not included in the kit. Perform synthesis using dntp Set (#R0181). 10 & Incubate the mixture at 42 C for 60min (incubate at 25 C for 10min and fi nally at 42 C for 60min if the random hexamer primer is used). ' Stop the reaction by adding 5µl of 0.5M EDTA. ( If required, hydrolyse RNA by the addition of equal volume (25µl) of 0.6N NaOH and incubate at 70 C for 30min. ) Remove unincorporated dntps by chromatography on a Sephadex G-50 column. The high specifi c activity fi rst strand cdna (>10 7 dpm/µg) obtained using this method can be used as a hybridization probe in Southern analysis. Notes To achieve an even higher specifi c activity (over 10 8 dpm/µg) of cdna, add 100µCi of [α- 32 P]dATP to the reaction mixture. If the fi nal reaction volume is larger than 20µl, vacuum-evaporate 10µl of [α- 32 P]dATP (10mCi/ml) in a separate tube and then transfer the prepared reaction mixture into the tube (5 step). Then proceed with the synthesis further on. 4. Analysis of First Strand cdna Products Only radiolabeled cdna products can be quantitifi ed or analyzed by alkaline agarose gel electrophoresis. Determination of the Yield of First Strand cdna! Spot 1µl aliquotes of your sample on two Whatman DE-81 fi lters (1.5x1.5cm). " Dry the fi lters under a heat lamp. Keep one fi lter aside and use it directly for the determination of total radioactivity in the sample. # Wash the other fi lter three times for 5min in 10ml of 7.5% (w/v) Na 2 HPO 4 x12h 2 O for removal of unincorporated dntps; rinse with water, wash with acetone or 96% ethanol.
11 $ Dry the washed fi lter under a heat lamp. % Transfer and count both fi lters (unwashed and washed) in a radioactivity counter. & Use the following formula to calculate the yield of fi rst strand cdna: Incorporated radioactivity (washed fi lter) mol datp cdna yield (µg) = x in the x 4 x MW total radioactivity assay (unwashed fi lter) If the fi nal concentration of dntp is 1.0mM, then mol datp in the assay (20µl) is 2x10-8 mol. The quantity of the labeled datp is comparatively low, so it could be neglected. However, in the case of the high specifi c activity cdna synthesis (chapter 3), the fi nal concentration of unlabeled datp is 0.02mM. For calculation of mol datp in the assay, it is necessary to sum up the molar amounts of both unlabeled and labeled datp. MW is the average molecular weight of nucleotide, which is equal to 333x10 6 µg/mol. As all four dntp are involved in the reaction, MW is multiplied by 4. cdna yield (µg) cdna yield % = x 100% RNA template (µg) 12 For example: If the washed fi lter gives 4x10 4 dpm and the unwashed fi lter 1.8x10 6 dpm, then: 4x10 4 dpm cdna yield (µg) = x (2x10-8 mol datp) x 4 x (333x10 6 µg/mol) = 0.59µg 1.8x10 6 dpm 0.59µg cdna yield % = x 100% = 59% 1µg Alkaline Agarose Gel Electrophoretic Analysis The fi rst strand cdna synthesis products labeled with [ 32 P] can be analyzed by electrophoresis in an alkaline agarose gel. Likewise, the synthesis products and the DNA marker used should also be labeled with [ 32 P].! Prepare 1.4% agarose gel in 30mM NaCl, 2mM EDTA and equilibrate for at least 1h in alkaline electrophoresis buffer (30mM NaOH, 2mM EDTA). " Transfer an aliquote (1x10 5 dpm) of the sample into a separate tube and dilute with water to 5µl. Add 5µl of 2x loading buffer (60mM NaOH, 2mM EDTA, 6% Ficoll 400, 0.05% bromophenol blue). The labeled DNA marker should be diluted with the same 2x loading buffer. Note. 2x loading buffer should be stored at -20 C or dye should be added just prior to use. # Load the samples and run the alkaline agarose gel at 5V/cm until the dye has migrated approximately two thirds of the length of the gel.
13 $ Soak the gel in 7% trichloroacetic acid (TCA) at room temperature for 30min. % Dry the gel under vacuum on a gel dryer, or dry it for 1-2 hours under layers of paper towels weighted with a glass plate. & Cover the dried gel with a plastic wrap and expose the gel to an X-ray fi lm overnight at room temperature. CONTROL SYNTHESIS 1.1 kb RNA with a 3 -poly(a) tail is provided as a control. $ Prepare the following reaction mixture in a tube on ice: control RNA (0.5µg/µl) 2µl primer: primer (0.5µg/µl) 1µl or random hexamer primer (0.2µg/µl) 1µl or control (sequence-specifi c) primer (10pmol) 2µl DEPC-treated water to 11µl Mix gently and spin down for 3-5sec. in a microcentrifuge. $ Incubate the mixture at 70 C for 5min, chill on ice and collect drops by brief centrifugation. $ Place the tube on ice and add the following components in the indicated order: 5x reaction buffer 4µl 10mM dntp mix 2µl RiboLock Ribonuclease Inhibitor (20u/µl) 1µl Mix gently and collect drops by brief centrifugation. $ Incubate at 37 C for 5min (at 25 C for 5min if the random hexamer primer is used). $ Add: [α- 32 P]dATP (10mCi/ml) 1µl RevertAid M-MuLV Reverse Transcriptase (200u/µl) 1µl Final volume 20µl 14 & Incubate the mixture at 42 C for 60min (incubate at 25 C for 10min and fi nally at 42 C for 60min if the random hexamer primer is used). ' Stop the reaction by adding 1µl of 0.5M EDTA and keep on ice. ( Analyze the products (see chapter 4). Notes 1. The yield of fi rst strand cdna is usually more than 50% when using control RNA. 2. If the control synthesis is carried out with either or the control (sequence-specifi c) primer a clear 1.1 kb band is observed. In the case of hexamer primer several shorter bands are usually observed.
15 TROUBLESHOOTING Low yield or no detectable product are two clear indications of a failed reverse transcriptase reaction. Possible cause Checking Remedy Degraded DNA template. Check integrity of RNA template in glyoxal or formaldehyde gel electrophoresis. Control RNA should be visible in the gel as a bright 1.1kb length band. RNase contamination in the reaction mixture. RevertAid M-MuLV Reverse Transcriptase inhibitors (SDS, EDTA, guanidine salts, phosphate, pyrophosphate, polyamines, spermine, spermidine). Perform the control reaction and analyze the products obtained (see Control Synthesis). Perform control synthesis with RNA sample mixed up with 1µg of control RNA. Synthesis yield should be more than 50% and a bright additional band of 1.1kb should be visible on agarose gel if synthesis has been carried out with or control primer. Working with the sample of RNA, be careful not to contaminate it with RNases. Store template RNA as well as control RNA at -70 C, avoid multiple freezing/thawing of the sample and keep it on ice after thawing. Prepare a reaction mixture under aseptic conditions, wear gloves at all time, treat all plastic ware in contact with samples with DEPC. Be careful not to contaminate the solutions. Precipitate RNA sample with 96% ethanol and wash with 75% ethanol (DEPC treated water should be used to prepare ethanol solution). Do not add inhibitors to the reactions mixture. 16 Possible cause Checking Remedy Unsuitable primer. Perform synthesis with other primer and analyze the products. Sequence-specifi c primer should be complementary to the 3 -end of RNA. If the RNA template contains transcriptional pauses, perform synthesis with a random hexamer primer. Procedural error in the fi rst strand cdna synthesis. Repeat cdna synthesis. QUALITY CONTROL All components of the kit are tested in a standard fi rst strand cdna synthesis reaction using a polyadenylated RNA transcript as a control template (with specifi c control primer, and random hexamer). Quality authorized by: Birute Gagiliene
17 References 1. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual (2nd Ed.) Cold Spring Harbor University Press, Cold spring Harbor, NY, 1989. 2. Ausubel, F.M. et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, 1999. 3. Gerarrd, G.F., D Alessio, J.M., Meth. in Mol. Biol., V. 16, 73-93, 1993. Trademarks Elugent is a registered trademark of Calbiochem Novabiochem, Inc. Ficoll is a registered trademark of Pharmacia Biotech, Inc. Milli-Q is a registered trademark of Millipore Corporation. Sephadex is a registered trademark of Pharmacia Biotech, Inc. Triton is a registered trademark of Rohm & Haas, Inc. Whatman is a registered trademark of Whatman Ltd. RevertAid, RiboLock are the trademarks of Fermentas. PRODUCT USE LIMITATION. This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to www.fermentas.com 4 Revised 05.2004