Protocol INTRODUCTION MATERIALS. Competitive RT-PCR: Estimation of Copy Number

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1 1 of 6 4/29/2009 3:29 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi: /pdb.prot4111 Protocol Competitive RT-PCR: Estimation of Copy Number Renée M. Horner This protocol was adapted from "The Use of Internal Controls for Relative and Competitive RT-PCR," contributed by René M. Horner, Chapter 13, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, INTRODUCTION In this protocol, sample and competitor RNAs (previously synthesized as described in Preparation of Competitor RNA for Competitive RT-PCR) are reverse transcribed (separately) in a pilot experiment. A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. This procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor. The experiment includes controls for sample-to-sample variations in RT efficiency. The amplification protocol described here is designed for six reactions (with 10% excess). This will be adequate for one sample using four competitor dilutions, one no-competitor control, and one no-template control. MATERIALS Reagents Taq DNA polymerase (5 units/µl) 10X RT-PCR Buffer for relative PCR Competitor RNA (contains [ - 32 P]ATP, see radioactive substances) 10 9 copies (as prepared in Preparation of Competitor RNA for Competitive RT-PCR) DEPC-treated H 2 O dntp solution (10 mm, contains all four dntps) Ethidium bromide Gene-specific forward primer (5 µm) Gene-specific reverse primer (5 µm) MMLV reverse transcriptase ( units/µl) Random primers (50 µm) SUPERaseIN (20 units/µl) (Ambion) Total RNA, 1-5 µg Equipment Thin-walled PCR tubes Programmable thermal cycler

2 2 of 6 4/29/2009 3:29 PM Equipment for agarose gel electrophoresis (see Agarose Gel Electrophoresis) Heating blocks, preset to 42 C, 70 C, and 92 C METHOD Single-Strand cdna Synthesis 1. Add 2 µl of 50 µm random primers to thin-walled PCR tubes on ice. Prepare one tube for the RNA sample and one tube for the competitor RNA. 2. Add 1-5 µg of total RNA to the sample tube (maximum volume = 10 µl) and 10 9 copies of competitor RNA to the other tube (maximum volume = 10 µl). 3. Add 4 µl of 10 mm dntp solution to each tube and bring the volume to 16 µl with DEPC-treated H 2 O. 4. Incubate for 10 minutes at 70 C to denature secondary structure, then immediately place the tubes on ice. 5. To each tube, add 2 µl of 10X RT-PCR buffer, 1 µl of 20 units/µl SUPERaseIN, and 1 µl of units/µl MMLV reverse transcriptase. 6. Incubate for one hour at 42 C and then inactivate the reverse transcriptase by incubating for 10 minutes at 95 C. 7. Proceed directly to Step 8, or store the cdna products at -20 C. (See Troubleshooting) PCR Amplification 8. Prepare the following PCR master mix: 10X RT-PCR buffer 35 µl 10 mm dntp mix 28 µl 5 M gene-specific forward primer 14 µl 5 M gene specific reverse primer 14 µl 5 units/µl Taq polymerase 1.75 µl DEPC-treated H 2 O µl 9. Aliquot 47 µl of the PCR master mix into each of 6 thin-walled PCR tubes labeled 1-6. Keep the tubes on ice. 10. Add 2 µl of the sample cdna prepared above (Step 7) to tubes Serial-dilute the competitor cdnas prepared above (Step 7) as follows: Undiluted: 1 µl of competitor cdna = 5 x 10 7 copies/µl 10-2 dilution: dilute 1 µl of competitor cdna into 99 µl of TE = 5 x 10 5 copies/µl 10-4 dilution: dilute 1 µl of 10 2 > dilution into 99 l of TE = 5 x 10 3 copies/µl 10-6 dilution: dilute 1 µl of 10 4 dilution into 99 l of TE = 5 x 10 1 copies/µl 12. Add 1 µl of the undiluted competitor cdna to tube 1, corresponding to 5 x 10 7 copies. 13. Add 1 µl of the 10 2 dilution of competitor to tube 2, corresponding to 5 x 10 5 copies.

3 3 of 6 4/29/2009 3:29 PM 14. Add 1 µl of the 10 4 dilution of competitor to tube 3, corresponding to 5 x 10 3 copies. 15. Add 1 µl of the 10 6 dilution of competitor to tube 4, corresponding to 5 x 10 1 copies. 16. Add 1 µl of H 2 O to tube 5, for a no-competitor control. 17. Add 3 µl of H 2 O to tube 6, for a no-template control. 18. Carry out PCR using the following parameters: Cycle number Denaturation Annealing Polymerization C for 30 sec Annealing temperature for 30 sec 72 C for 30 sec 19. Analyze 5 µl of each reaction on a 2-2.5% agarose gel containing 1 µg/ml ethidium bromide (see Agarose Gel Electrophoresis). (See Troubleshooting) Two bands should be visible in each sample, one corresponding to the sample cdna amplicon, the other, the competitor cdna amplicon. Because the competitor is designed to have the same reaction kinetics as the gene of interest and is co-amplified with the gene of interest, the amount of product from each reaction can be compared directly. Equivalent band intensities indicate equal amounts of sample message and competitor transcript. Results are expressed in copy numbers or mass of transcript. 20. To control for variations in the efficiency of reverse-transcribing the competitor and the endogenous transcript, perform a final RT-PCR experiment in which the sample RNA and the competitor RNA are reversetranscribed in the same tube. Use several amounts of competitor RNA that are close to the amount indicated by the initial RT-PCR experiments, and perform the reverse transcription and amplification steps as described above. As before, the amount of competitor RNA that produces a signal of equal intensity to that produced by the endogenous message represents the amount of endogenous message in the sample. (See Troubleshooting) TROUBLESHOOTING Problem: Unsatisfactory RT-PCR [Step 7] Solution: Low yield in RT-PCR can be caused by degraded or impure RNA. Inhibitors of reverse transcriptase include guanidinium, proteinase K, and alcohol, which can be carried over from the RNA isolation procedure. Typically, the contaminant can be removed by phenol:chloroform extraction, reprecipitation, and washing of the pellet. Problem: Low yields or non-specific products after PCR [Steps 19 and 20] Solution: Poor primer design or nonoptimized PCR conditions can lead to poor yield and nonspecific product formation. The magnesium concentration can be varied from 1.5 to 4 mm to optimize the PCR chemistry. The annealing temperature of the PCR can be raised to alleviate nonspecific priming. Additionally, the hold times used in the PCR cycling may also have to be adjusted to obtain the highest possible yields. Thermal cyclers with short ramp times may need longer hold times at each temperature. A PCR signal from the no-reverse-transcriptase controls could signify genomic DNA contamination. In this case, the RNA samples should be digested with RNase-free DNase (Ambion) for 15 minutes at 37 C, followed by the addition of EDTA to 2.5 mm and incubation at 70 C to deactivate

4 4 of 6 4/29/2009 3:29 PM the enzyme. False positives in the no-template controls can signify contamination of one of the reagents or laboratory equipment by the target. Careful laboratory practices are essential; for example, PCRs should not be stored or used in the same location where PCR setup occurs. DNAZap (Ambion) can be used to eliminate nucleic acid contamination on laboratory surfaces such as PCR machines, micropipettors, and racks. See Chapter 1 for information about contamination control. ACKNOWLEDGMENTS I thank Ellen Prediger, Lori Martin, and Heath Thomas for supplying materials used in this protocol. I also thank James W. Horner II for critical reading of the manuscript. REFERENCES Kwok S. and Higuchi R Avoiding false positives with PCR. Nature 339: [Medline] Caution Ethidium bromide Ethidium bromide is a powerful mutagen and is toxic. Consult the local institutional safety officer for specific handling and disposal procedures. Avoid breathing the dust. Wear appropriate gloves when working with solutions that contain this dye. Caution Radioactive substances Radioactive substances: When planning an experiment that involves the use of radioactivity, consider the physicochemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and use only as much as needed. Always wear appropriate gloves, lab coat, and safety goggles when handling radioactive material. X rays and gamma rays are electromagnetic waves of very short wavelengths either generated by technical devices or emitted by radioactive materials. They might be emitted isotropically from the source or may be focused into a beam. Their potential dangers depend on the time period of exposure, the intensity experienced, and the wavelengths used. Be aware that appropriate shielding is usually made of lead or other similar material. The thickness of the shielding is determined by the energy(s) of the X rays or gamma rays. Consult the local safety office for further guidance in the appropriate use and disposal of radioactive materials. Always monitor thoroughly after using radioisotopes. A convenient calculator to perform routine radioactivity calculations can be found at: 10X RT-PCR buffer for relative PCR 100 mm Tris-HCl, ph mm KCl

5 5 of 6 4/29/2009 3:29 PM 15 mm MgCl 2 Use RNase-free H 2 O when preparing this buffer. Diethyl pyrocarbonate (DEPC)-treated H 2 O Distilled H 2 O Diethyl pyrocarbonate (DEPC) Add 1 ml of fresh DEPC to 1 L of H 2 O. Shake well to disperse the DEPC through the H 2 O. Incubate at 37 C for at least 12 h and/or autoclave at 15 psi on liquid cycle for 20 min to inactivate the remaining DEPC. Ethidium bromide Add 1 g of ethidium bromide to 100 ml of H 2 O. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperature. dntp solution Dissolve each dntp (deoxyribonucleoside triphosphates) in H 2 O at an approximate concentration of 100 mm. Use 0.05 M Tris base and a micropipette to adjust the ph of each of the solutions to 7.0 (use ph paper to check the ph). Dilute an aliquot of the neutralized dntp appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dntp. Dilute the solutions with H 2 O to a final concentration of 50 mm dntp. Store each separately at -70 C in small aliquots. For polymerase chain reactions (PCRs), adjust the dntp solution to ph 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dntps require no adjustment. Base wavelength (nm) Extinction Coefficient (E) (M -1 cm -1 ) A x 10 4 G x 10 4 C x 10 3 T x 10 3 For a cuvette with a path length of 1 cm, absorbance = EM. 100 mm stock solutions of each dntp are commercially available (Pharmacia). Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials

6 6 of 6 4/29/2009 3:29 PM used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission. CiteULike Connotea Del.icio.us Digg Reddit Technorati What's this?

Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis.

Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis. 1 of 6 4/29/2009 2:49 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot3815 Protocol Detection of Mutations by Single-strand Conformational Polymorphism Joseph Sambrook and David W. Russell