[Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR)].

Abstract

Due to its extraordinarily high sensitivity, polymerase chain reaction (PCR) is widely used for amplifying cDNA copies of low abundance mRNA. However, quantitation is unreliable because the amount of PCR product increases exponentially with each cycle of amplification; therefore, minute differences in any of the variables that affect the efficiency of amplification can dramatically alter product yield. Coamplification with a different reporter gene product is a semiquantitative mRNA quantitation method. Thus, we constructed an internal standard with a defined deletion fragment from the target cDNA, and used the same primers to coamplify the unknown and the competitor, allowing us to quantify the amount of specific target cDNA available. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube-to-tube or sample-to-sample variations in the kinetics of the RT reaction. For RNA quantitation, Northern blots are widely used. However, the Northern blot technique requires at least 10mg of total RNA for semiquantitation while QC-PCR requires only 1mg of total RNA and is useful when only small amounts of tissue are available. QC-PCR is a simple, and inexpensive method in which competitive PCR is used for highly accurate quantitation of mRNA and DNA from a small number of cells.