Quantitation of mRNA by the Polymerase Chain Reaction

Abstract

Polymerase chain reaction (PCR) is a powerful method for analyzing RNAs from a smaller number of cells or for characterizing very rare mRNA species [1–5]. However, in most instances, the PCR technique has only provided qualitative results. The availabibity of an accurate quantatative PCR method should provide valuable additional information for these studies. It has been difficult to quantitate the absolute amount of specific mRNA without an internal standard of known concentration. Because PCR amplification is an exponential process, minute differences in any of the variables that affect the efficiency could lead to large differences in the yield of PCR product. This problem has been addressed by co-amplification of the mRNA of interest with unrelated template as an internal standard [6–8]. However, this approach provides only comparative data, in part because of differences in efficiency between the primer pairs for the standard and the target mRNAs. We have developed a technique in which synthetic RNA is used as an internal standard for quantitating the amount of specific mRNA by PCR [9]. This technique involves the co-amplification of a target mRNA with the internal standard. This standard uses the same primer sequences as the target mRNA but yields a PCR product of a different size. The two PCR products can then be seperated by gel electrophoresis.