Competitive RT-PCR: Estimation of Copy Number.

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.