Quantitative mRNA-PCR for expression analysis of low-abundance transcripts

Reverse transcription PCR (RT-PCR) has been proven to be a useful method in the analysis of gene expression, especially for detecting low abundance mRNA transcripts. However, quantitation by RT-PCR can be difficult due to different reverse transcription and PCR amplification efficiencies and sample-to-sample and tube-to-tube variations. We have used synthetic RNA that contains the same primer sequences as the target mRNA, as an internal standard, to improve the accuracy of RT-PCR quantitation. Products generated from the target gene by RT-PCR differ in size from those of the synthetic internal standard. Using the ratios of two species of PCR products as a quantitation index, sample-to-sample and tube-to-tube variations can be eliminated. The method we present here can be widely applied to analyzing gene expression in different tissues or cell types with minimal effort to produce standard curves.