SMART mrna Amplification Kit. User Manual. User Manual

Transcription

1 User Manual SMART mrna Amplification Kit User Manual United States/Canada Asia Pacific Europe +33.(0) Japan +81.(0) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Ave. Mountain View, CA Technical Support (US) tech@clontech.com Cat. No PT (PR872566) Published January 2010

2 Table of Contents I. Introduction & Protocol Overview 3 II. List of Components 5 III. Additional Materials Required 6 IV. General Considerations 7 V. SMART mrna Amplification Procedure 8 A. First-Strand cdna Synthesis 8 B. Primer Extension (Second-Strand cdna Synthesis) 9 C. NucleoSpin Extraction 9 D. T7 Transcription 10 E. NucleoSpin RNA II Purification 11 VI. Troubleshooting Guide 12 VII. References 13 Appendix A: Assessing Yield and Purity of RNA 14 Appendix B: Analyzing First-Strand cdna 16 List of Figures and Tables Figure 1. Flow chart of the SMART mrna amplification method 4 Figure 2. Analysis of first-strand cdna synthesized for SMART mrna amplification 17 Table I. PCR cycling parameters to detect proper template switching 18 Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

3 I. Introduction & Protocol Overview SMART mrna Amplification Kit User Manual The SMART mrna Amplification Kit provides a fast and easy method for generating large amounts of mrna from limited starting material. This kit combines our SMART technology with T7 RNA polymerase transcription. You can synthesize high-quality sense RNA (srna) for use in array probe generation, quantitative RT-PCR, cdna cloning, or other applications, from as little as 100 ng of total RNA. Typical yields of µg of srna are achieved by following this User Manual. SMART mrna amplification technology SMART mrna amplification begins with the generation of a double-stranded cdna template. As a first step, nanogram quantities of total RNA and a modified oligo(dt) primer (the cdna Synthesis Primer II A) are used to initiate first-strand cdna synthesis (Figure 1). When the reverse transcriptase (RT) reaches the end of the mrna, the enzyme s terminal transferase activity adds a few additional nucleotides, primarily deoxycytidine, to the 3' end of the cdna. The SMART T7 Oligonucleotide, which has an oligo(g) sequence at its 3' end, base-pairs with the deoxycytidine stretch, creating an extended template. RT then switches templates and continues replicating to the end of the oligonucleotide (Chenchik et al., 1998). The resulting full-length, singlestranded (ss) cdna contains sequences that are complementary to the SMART T7 Oligonucleotide. The SMART T7 anchor sequence is then used for primer extension to generate double-stranded cdna. Finally, sense RNA is transcribed in vitro using T7 RNA polymerase. The T7 RNA polymerase has high specificity for its promoter, and once transcription is initiated elongation is very rapid. Reinitiation and elongation continue until the supply of ribonucleotides is exhausted, resulting in linear amplification of mrna. Advantage 2 PCR Kit We strongly recommend use of the Advantage 2 PCR Kits (Cat. Nos & ) for PCR amplification. These kits include the Advantage 2 Polymerase Mix, which has been specially formulated for efficient, accurate, and convenient amplification of cdna templates by long-distance PCR (LD PCR; Barnes, 1994). The Polymerase Mix is comprised of TITANIUM Taq DNA Polymerase a nuclease-deficient N-terminal deletion of Taq DNA polymerase plus TaqStart Antibody to provide automatic hot-start PCR (Kellogg et al., 1994) and a minor amount of a proofreading polymerase. This combination allows you to efficiently amplify full-length cdnas with a significantly lower error rate than that of conventional PCR (Barnes, 1994). Protocol No. PT Clontech Laboratories, Inc. Version No. PR

4 I. Introduction & Protocol Overview continued T7 GGG SMART T7 Oligonucleotide RNA polya 3' CDS primer II A First-strand synthesis by RT T7 GGG polya dc tailing by RT Single step T7 GGG CCC polya Template switching and extension by RT T7 GGG CCC polya Primer extension T7 Double-stranded cdna In vitro transcription Sense RNA (srna) AAA 3' AAA 3' AAA 3' AAA 3' AAA 3' Figure 1. Flow chart of the SMART mrna amplification method. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

5 II. List of Components SMART mrna Amplification Kit User Manual Store the SMART T7 Oligonucleotide and Control Total RNA at 70 C. Store the NucleoSpin Extraction and NucleoSpin RNA II Kits at room temperature. Store all other components at 20 C. The following reagents are suitable for 10 reactions. Box 1: 10 µl SMART T7 Oligonucleotide (10 µm) -ACTCTAATACGACTCACTATAGGGAGAGGGCGGG-3' 5 µl Control Total RNA (Human Placenta, 1 µg/µl) Box 2: 10 µl cdna Synthesis (CDS) Primer II A (12 µm) -AAGCAGTGGTATCAACGCAGAGTACT (30) VN-3' (N = A, C, G, or T; V = A, G, or C) 200 µl 5X First-Strand Buffer 20 µl DTT (100 mm) 50 µl 50X dntp Mix (10 mm each datp, dgtp, dctp, and dttp) 30 µl T7 Extension Primer (10 µm) -GCTCTAATACGACTCACTATAGG-3' 10 µl RNase H (10 U/µl) 200 µl 10X T7 Transcription Buffer 70 µl 3X rntp Mix 10 µl T7 RNA Polymerase (1,000 U/µl) 30 µl PCR Primer II A (12 µm) * -AAGCAGTGGTATCAACGCAGAGT-3' 30 µl Linear Acrylamide (5 µg/µl) 340 µl Sodium Acetate (3 M) * This component is provided for troubleshooting purposes only. See Appendix B for more information. Box 3: NucleoSpin RNA II Purification Kit Box 4: NucleoSpin Extract II Kit (10 preps) Protocol No. PT Clontech Laboratories, Inc. Version No. PR

6 III. Additional Materials Required The following materials are required but not supplied: MMLV Reverse Transcriptase We recommend SMART MMLV Reverse Transcriptase (Cat. Nos , & ). Please see Addendum PT for details. Advantage 2 PCR Kit (Cat. Nos & ) RNase Inhibitor (20 U/µl) We recommend Ambion s SUPERase In RNase inhibitor (Cat. No. 2696). Ethanol Buffer for measuring O.D. (see Appendix A) 10 mm Tris (ph 7.5) 0.1 mm EDTA (ph 7.5) Store at 20 C. Quartz cuvettes UV/Vis spectrophotometer For a complete listing of all Clontech Products, please visit Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

7 IV. General Considerations SMART mrna Amplification Kit User Manual The success of your experiment depends on the quality of your starting sample of total RNA. For best results we strongly recommend that you use the NucleoSpin RNA II Kit (included). Alternatively, you may wish to use one of our already-made Premium Total RNAs (visit our web site at There are also several procedures available for RNA isolation (Chomczynski & Sacchi, 1987; Farrell, 1993; Sambrook et al., 2001). Before you begin first-strand synthesis, we strongly recommend that you check the integrity of your RNA. For determining the integrity of human total RNA samples, we recommend that you use the Clontech RNA/cDNA Quality Assay (Cat. No ). Alternatively, you can check RNA quality by electrophoresing a sample on a formaldehyde/agarose/etbr gel, if you have sufficient RNA (see Appendix A). For mammalian total RNA, you should observe two bright bands at approximately 4.5 and 1.9 kb; these bands represent 28S and 18S ribosomal RNA. The ratio of intensities of these bands should be :1. For more information, see Sambrook et al. (2001). Purification of srna is performed by following the protocol in this User Manual. Do not follow the protocol in the NucleoSpin User Manual. Wear gloves throughout the procedure to protect your RNA and cdna samples from degradation by nucleases. The first time you use this kit, you should perform cdna synthesis with the Control Total RNA provided in the kit, in parallel with your experimental sample. Performing this control at least once will verify that all components are working properly, and will also help you troubleshoot any problems that may arise. The cycling parameters in this protocol have been optimized using a hotlid thermal cycler. Optimal parameters may vary with different thermal cyclers and templates. To resuspend pellets and mix reactions, gently pipet them up and down and centrifuge the tube briefly to deposit contents at the bottom. Add enzymes to reaction mixtures last, and thoroughly incorporate the enzyme by gently pipetting the reaction mixture up and down. Do not increase the amount of enzyme added or concentration of RNA in the reactions. The amounts and concentrations have been carefully optimized. Protocol No. PT Clontech Laboratories, Inc. Version No. PR

8 V. SMART mrna Amplification Procedure PLEASE READ ENTIRE PROTOCOL BEFORE STARTING. A. First-Strand cdna Synthesis 1. Preheat a thermal cycler to 70 C. 2. For each sample and control, combine the following reagents in a sterile 0.5 ml reaction tube: 1 3 µl RNA sample (0.1 5 µg)* 1 µl CDS Primer II A (12 µm) x µl Deionized H 2 O 4.25 µl Total volume * For the control synthesis, add 1 µl of Control Total RNA (Human Placenta, 1 µg/µl). 3. Mix contents and spin the tube briefly in a microcentrifuge. 4. Incubate the tube at 70 C for 3 min, then reduce the temperature to 42 C for 2 min. 5. Prepare a Master Mix for all reaction tubes, plus one additional tube. a. Combine the following components in the order shown: per rxn 2 µl 5X First-Strand Buffer 0.5 µl DTT (100 mm) 0.25 µl RNase inhibitor 1 µl SMART T7 Oligonucleotide (10 µm) 1 µl 50X dntp Mix (10mM each datp, dgtp, dctp, and dttp) 1 µl MMLV Reverse Transcriptase 5.75 µl Total volume Add enzyme to Master Mix just prior to use. Please see Addendum PT for details on the choice of RT enzyme. b. Mix well by vortexing and spin the tube briefly in a microcentrifuge. 6. Aliquot 5.75 µl of the Master Mix into each reaction tube. Use a fresh pipet tip for each tube, and mix thoroughly by pipetting. Spin the tube briefly in a microcentrifuge. Immediately return tube to the thermal cycler. 7. Incubate all reaction tubes at 42 C for 1.5 hr. 8. Terminate the reaction by heating at 68 C for 10 min. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

9 V. SMART mrna Amplification continued SMART mrna Amplification Kit User Manual B. Primer Extension (Second-Strand cdna Synthesis) 1. Prepare a Master Mix for all reaction tubes, plus one additional tube. Combine the following components in the order shown: per rxn 73 µl Deionized H 2 O 10 µl 10X Advantage 2 PCR Buffer 2 µl 50X dntp Mix (10mM each datp, dgtp, dctp, and dttp) 2 µl T7 Extension Primer (10 µm) 1 µl RNase H (10 U/µl) 2 µl 50X Advantage 2 Polymerase Mix 90 µl Total volume 2. Mix well by vortexing and spin the tube briefly in a microcentrifuge. 3. Aliquot 90 µl of the Master Mix into each labeled 0.5 ml reaction tube from Step A Cap the tube, and place it in the preheated thermal cycler. 5. Commence thermal cycling using the following program*: 37 C 15 min 95 C 2 min 60 C 1 min 68 C 10 min * Optimal parameters may vary with different thermal cyclers and different templates. 6. Proceed to Section C to purify your double-stranded cdna. C. NucleoSpin Extraction Use the provided NucleoSpin Extract II Kit and follow the procedure described below to purify your double-stranded cdna prior to performing in vitro transcription. Alternatively, you may purify your samples using QIAGEN's MinElute Reaction Cleanup Kit (Cat. No ). Important: Before beginning, ensure that 95% ethanol has been added to Buffer NT3 according to the instructions on the bottle. 1. Mix 2 volumes of buffer NT with 1 volume of sample (e.g. 200 µl NT and 100 µl PCR reaction mix). Note: For sample volumes < 50 µl adjust the volume of the reaction mix to 50 µl using TE buffer (ph 7.5). 2. Place a NucleoSpin Extract II column into a 2 ml collecting tube and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin Extract II column back into the collecting tube. Protocol No. PT Clontech Laboratories, Inc. Version No. PR

10 V. SMART mrna Amplification continued 3. Add 600 µl buffer NT3. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin Extract II column back into the collecting tube. 4. Centrifuge for 2 min at 11,000 x g to remove buffer NT3 quantitatively. Make sure the spin column doesn t come in contact with the flow-through while removing it from the centrifuge and the collecting tube. 5. Place the NucleoSpin column in a clean 1.5 ml microcentrifuge tube. Add 50 µl of Buffer NE, and let stand 1 min. Centrifuge at maximum speed for 1 min. Note: If you do not want to subject your sample to ethanol precipitation (Steps 8 12), elute using 25 µl deionized water. Proceed from this point by using 9 µl of this eluate in Section V.D. 6. To ensure complete elution of cdna, add a second 50 µl of Buffer NE to the NucleoSpin column. Let stand 1 min, and centrifuge at maximum speed for 1 min. Discard the NucleoSpin column and centrifuge the eluted cdna at maximum speed for an additional 3 min. Transfer the supernatant to a new tube. 7. Add 3 µl Linear Acrylamide, 10 µl Sodium Acetate (3 M), and 250 µl 100% ethanol; vortex. 8. Place tube on dry ice for 15 min to precipitate the cdna. Note: Alternatively, you may precipitate your cdna by placing the tube in a 20 C freezer for 2 hr (or overnight). 9. Spin tube at maximum speed in a microcentrifuge for 20 min. 10. Carefully pipet off supernatant, and wash pellet once in 100 µl of 70% ethanol for 10 min. 11. Dissolve pellet in 9 µl deionized H 2 O. D. T7 Transcription 1. Prepare a Transcription Master Mix for all reaction tubes, plus one additional tube. Combine the following components at room temperature, in the order shown: per rxn 2 µl 10X T7 Transcription Buffer 7 µl 3X rntp Mix 1 µl RNase inhibitor 1 µl T7 RNA Polymerase (1,000 U/µl) 11 µl Total volume 2. Mix well by vortexing and spin the tube briefly in a microcentrifuge. 3. Aliquot 11 µl of the Transcription Master Mix into each tube from Step C.11. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

11 V. SMART mrna Amplification continued SMART mrna Amplification Kit User Manual Note: If you are using purified cdna that has not been ethanol precipitated (see Step C.6 Note), aliquot 9 µl of eluate into a clean 1.5 ml microcentrifuge tube. Add 11 µl of the Transcription Master Mix into each tube. 4. Mix well by vortexing and spin the tube briefly in a microcentrifuge. 5. Incubate the tube at 37 C for 12 hr. E. NucleoSpin RNA II Purification To purify the mrna from unincorporated ribonucleotides and small (<0.1 kb) cdna and RNA fragments, use the provided NucleoSpin RNA II Kit and follow the procedure described below. Do not follow the protocol outlined in the NucleoSpin User Manual. Important: Before beginning, ensure that 95% ethanol has been added to Buffer RA3 according to the instructions on the bottle. 1. Add 300 µl of Buffer RA1 to each tube from Step D.5. Mix well by pipetting. 2. Add 240 µl 100% ethanol to each tube. Mix well. 3. Place the NucleoSpin column (blue) in a 2 ml Collection Tube and load the sample into the column. Centrifuge at 8,000 x g for 60 sec. Discard the flowthrough. 4. Add 750 µl of Buffer RA3 to the NucleoSpin column. Centrifuge at 14,000 x g for 1 min. Discard the flowthrough, and place the NucleoSpin column back in the 2 ml Collection Tube. 5. Repeat Step 4 two times, using 250 µl of Buffer RA3 each time. 6. Centrifuge at 14,000 x g for 1 min to completely remove any residual wash buffer and dry the column filter. Place the NucleoSpin column into a 1.5 ml microcentrifuge tube (provided). 7. Elute the RNA by adding Nuclease-free Water, allowing the filter to soak for 2 min, and centrifuging at maximum speed for 1 min. We recommend eluting in two steps using 35 µl for the first elution and 20 µl for the second elution. For concentrated RNA samples, elute with 40 µl of Nuclease-free Water. After elution, discard the NucleoSpin column and centrifuge the eluted RNA at maximum speed for an additional 3 min. Transfer the supernatant to a new tube. Estimate the yield by UV spectroscopy and gel electrophoresis (see Appendix A). Protocol No. PT Clontech Laboratories, Inc. Version No. PR

12 VI. Troubleshooting Guide A. Poor yield from the Control Total RNA 1. Proper template switching, which is essential to the SMART technology, requires the use of an MMLV reverse transcriptase. To determine whether template switching occurred, a PCR protocol is provided in Appendix B for analyzing the first-strand cdna reaction. 2. RNAs may have degraded during storage and/or first-strand synthesis. Poor quality RNA starting material will reduce the ability to obtain full-length cdnas and consequently, full-length srnas. RNA samples must be stored at 70 C. Your working area, equipment, and solutions must be free of contamination by RNase (we recommend using RNase Blaster, Cat. No ). For best results, freeze cells/tissue immediately following harvest in Buffer RA1 with an RNase inhibitor, then use the NucleoSpin RNA II Kit to isolate RNA (see Related Products for ordering information). 3. You may have made an error during the procedure, such as using a suboptimal incubation temperature or omitting an essential component. Carefully check the protocol and repeat the experiment with your sample and the control RNA. B. Poor yield from your experimental RNA sample If the reaction with the Control Total RNA was successful, but your experiment failed, your experimental RNA sample may be too dilute or degraded, or may contain impurities that inhibit first-strand synthesis. If you have not already done so, electrophorese a sample of your RNA on a formaldehyde/agarose/etbr gel to determine its concentration and analyze its quality (see Appendix A for more details). 1. The concentration of your experimental RNA is low, but the quality is good. Repeat the experiment using more RNA. 2. Your experimental RNA has been partially degraded (by contaminating RNases) before or during first-strand synthesis. Repeat the experiment using a fresh lot or preparation of RNA. Check the stability of your RNA by incubating a small sample in water for 2 hr at 42 C. Then, electrophorese it on a formaldehyde/agarose/etbr gel alongside an unincubated sample. If the RNA is degraded during incubation, it will not yield good results in the first-strand synthesis. In this case, reisolate the RNA using a different technique, such as our NucleoSpin RNA II Kit (see Related Products for ordering information). Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

13 VI. Troubleshooting Guide continued SMART mrna Amplification Kit User Manual 3. Your experimental RNA sample contains impurities that inhibit cdna synthesis. In some cases, ethanol precipitation of your existing total RNA, followed by washing twice in 80% EtOH, may remove impurities. If this fails, reisolate the RNA using a different technique, such as our NucleoSpin RNA II Kit. VII. References Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91: Chenchik, A., Zhu, Y. Y., Diatchenko, L., Li, R., Hill, J. & Siebert, P. D. (1998) Generation and use of high-quality cdna from small amounts of total RNA by SMART PCR. In Gene Cloning and Analysis by RT-PCR (BioTechniques Books, MA), pp Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: Farrell, Jr., R. E. (1993) RNA Methodologies A Lab Guide for Isolation and Characterization (Academic Press, San Diego, CA). Kellogg, D. E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik, T., Siebert, P. & Chenchik, A. (1994) TaqStart Antibody: Hotstart PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16: Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY). Protocol No. PT Clontech Laboratories, Inc. Version No. PR

14 Appendix A: Assessing Yield and Purity of RNA Use the following protocol to determine the yield and quality of your RNA starting material and your purified srna. A. Determining A Thoroughly mix your RNA. Measure the entire RNA sample volume. 2. Transfer 2 5 µl of your RNA sample to a 1.5 ml tube. 3. Bring volume up to 400 µl with O.D. buffer (see Section III) and mix by pipetting. 4. Transfer contents to a 1 ml glass cuvette with a 1 cm path length. 5. Measure A 260 and A 280 using O.D. buffer as a reference blank. 6. Calculate RNA yield as follows: RNA constant for 1 cm path length: One A 260 unit of RNA = 40 µg/ml Total A 260 = (A 260 of dilute sample) x (dilution factor) Concentration (µg/ml) = (total A 260 ) x (40 µg/ml) Yield (µg) = (total sample volume) x (concentration) 7. Calculate the A 260 /A 280 ratio. Pure RNA exhibits a ratio of Example: The RNA sample volume was 0.5 ml. A 2 µl sample aliquot was diluted to 400 µl in O.D. buffer. The following spectrophotometric readings were taken: A 260 = 0.231; A 280 = Calculations: Total A 260 = (0.231) x (200) = 46.2 Concentration = (46.2) x (40) = 1,848 µg/ml RNA yield = (0.5 ml) x (1,848 µg/ml) = 924 µg Purity = 0.231/0.115 = 2.01 B. Preparing a 1% Denaturing Agarose Gel 1. Wash mini-gel box, gel tray (7 x 10 cm), and combs with deionized water. Equipment should be reserved for RNA work only. 2. Add 1 g of agarose to a 250 ml beaker containing a magnetic stirbar. 3. Add 82.5 ml of water. 4. Microwave for 2 min or until boiling. Perform the following steps in a fume hood: 5. Place bottle on a magnetic stir plate and stir slowly for 2 min to cool. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

15 Appendix A: Assessing Yield and Purity continued 6. While stirring, add 10 ml of 10X MOPS buffer and 7.5 ml of 12.3 M formaldehyde. 7. Continue stirring for 1 min; then pour onto gel tray. 8. Allow at least 1 hr for the gel to solidify at room temperature. Note: Do not use formaldehyde gels that have been stored longer than 24 hr. 9. Remove the gel comb and submerge the gel in gel box with 1X MOPS buffer. C. RNA Sample Preparation 1. Prepare RNA loading solution immediately before running the gel (for 6 10 samples): formaldehyde 45 µl formamide 45 µl 10X MOPS buffer 10 µl EtBr (10 mg/ml) 3.5 µl 0.1 M EDTA (ph 7.5) 1.5 µl bromophenol blue dye (in 50% glycerol) 8 µl 2. Add µl of RNA loading solution to 1 2 µg of total RNA; mix well. 3. Heat at 70 C for min. 4. Cool on ice 1 min, then load on gel. D. Gel Electrophoresis Guidelines 1. Run gel at 4 5 V/cm (equivalent to V on a mini-gel box). 2. Examine gel when dye has migrated 3 4 cm from the wells. E. Expected Results Amplified srna should appear as a distinct smear from kb. Total RNA from mammalian sources should appear as two bright bands (28S and 18S ribosomal RNA) at approximately 4.5 and 1.9 kb. The ratio of intensities of the 28S and 18S rrna bands should be :1. Lower ratios are indicative of degradation. You may also see additional bands or a smear lower than the 18S rrna band, including very small bands corresponding to 5S rrna and trna. Protocol No. PT Clontech Laboratories, Inc. Version No. PR

16 Appendix B: Analyzing First-Strand cdna Use the following protocol to determine whether template switching occurred during the first-strand cdna synthesis (Section V.A). This protocol analyzes your first-strand cdna reaction with the PCR Primer II A and the T7 Extension Primer in a PCR reaction using a series of amplification cycles. Figure 2 shows the characteristic result of proper template switching. 1. Preheat a thermal cycler to 95 C. 2. Prepare a Master Mix for all reaction tubes, plus one additional tube. Combine the following components in the order shown: per rxn 40 µl Deionized H 2 O 5 µl 10X Advantage 2 PCR Buffer 1 µl 50X dntp mix 1 µl PCR Primer II A 1 µl T7 Extension Primer 1 µl 50X Advantage 2 Polymerase Mix 49 µl Total volume 3. Mix well by vortexing and centrifuge the tube briefly in a microcentrifuge. 4. Aliquot 49 µl of the Master Mix into each reaction tube. 5. Aliquot 1 µl of each first-strand cdna (from Section V.A.8) into each reaction tube. 6. Mix contents by gently flicking the tubes. Centrifuge tubes briefly in a microcentrifuge. 7. Cap the tube, and place it in the preheated thermal cycler. 8. Commence thermal cycling using the following program: 95 C 1 min x cycles*: 95 C 15 sec 65 C 30 sec 68 C 3 min * Subject all tubes to 15 cycles. Then subject all tubes to additional PCR cycles, as described in Step 9 (below). 9. For each PCR tube, determine the optimal number of PCR cycles (see Figure 2): a. Transfer 5 µl from the 15 cycle PCR to a clean microcentrifuge tube (for agarose/etbr gel analysis). b. Run three additional cycles (for a total of 18) with the remaining 45 µl of the PCR mixture. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566

17 Appendix B: Analyzing First-Strand cdna continued M kb Figure 2. Analysis of first-strand cdna synthesized for SMART mrna amplification. 1 µl (1.0 µg) of the Control Total RNA provided in the kit was used as starting material in a first-strand cdna synthesis. 1 µl of the ss cdna then served as template for PCR-based second-strand synthesis using 15, 18, and 21 cycles. A 5 µl sample of the PCR product (i.e., ds cdna) was electrophoresed on a 1.2% agarose/etbr gel. Lane M: 1 kb DNA ladder size markers, 0.1 µg loaded. The arrow indicates the band at 900 bp typically seen for the Control Total RNA. For detecting sufficient template switching, the optimal number of PCR cycles for this experiment was 15. c. Transfer 5 µl from the 18 cycle PCR to a clean microcentrifuge tube (for agarose/etbr gel analysis). d. Run three additional cycles (for a total of 21) with the remaining 40 µl of PCR mixture. e. Transfer 5 µl from the 21 cycle PCR to a clean microcentrifuge tube (for agarose/etbr gel analysis). f. Run three additional cycles (for a total of 24) with the remaining 35 µl of PCR mixture. 10. Electrophorese 5 µl of each PCR reaction alongside 0.1 µg of 1 kb DNA size marker on a 1.2% agarose/etbr gel in 1X TAE buffer. Figure 2 shows a characteristic gel profile of ds cdna synthesized from Protocol No. PT Clontech Laboratories, Inc. Version No. PR

18 Appendix B: Analyzing First-Strand cdna continued the Control Total RNA. As a reference, Table I provides the optimal number of PCR cycles typically found for a given starting amount of total RNA used in the first-strand synthesis (from Section V.A). The optimal number of PCR cycles is the minimum number of cycles necessary to determine that template switching occurred during first-strand synthesis. table i: pcr cycling parameters to detect proper template switching Starting Amount of Total RNA (µg) Optimal Number of Cycles These parameters were developed using the Control Total RNA and an authorized hot-lid thermal cycler; optimal parameters may vary with different templates and thermal cyclers. Notice to Purchaser Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc. MinElute is a trademark of QIAGEN. NucleoSpin is a registered trademark of Macherey-Nagel GmbH & Co. SMART Technology is covered by U.S. Patent Nos. 5,962,271 and 5,962,272. For-Profit and Not- For-Profit purchasers of SMART Products are entitled to use the reagents for internal research. However, the following uses are expressly prohibited: (1) performing services for third parties; (2) identifying nucleic acid sequences to be included on nucleic acid arrays, blots, or in libraries or other cdna collections which are then sold to third parties. Reproduction, modification, reformulation, or resale of the reagents provided in SMART Products is not permitted. For information on licensing SMART Technology for commercial purposes, please contact a licensing representative by phone at or by at licensing@clontech.com. Clontech, the Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc., unless noted otherwise. Clontech is a Takara Bio Company Clontech Laboratories, Inc. Clontech Laboratories, Inc. Protocol No. PT Version No. PR872566