Quantitative Analysis of mRNA Levels in Xenopus Embryos by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Over the last few years, RT-PCR (1 ,2 ) has become a widely accepted method for quantitation of steady-state mRNA levels, particularly in Xenopus . Its unmatched sensitivity and swiftness allows for a high sample throughput with minimal amounts of starting material—considerable advantages over the conventional methods of Northern blotting or RNase protection. Initially, the use of RT-PCR for quantitative analysis was viewed skeptically. This was based on the concern that minor differences in the reaction conditions between samples would erratically influence the exponential rate of PCR amplification; therefore, results would be skewed a priori . This theoretical concern has turned out to be irrelevant for most applications.