Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR

The PCR has proven to be useful in the analysis of gene expression of specific mRNAs. Although PCR is able to detect rare mRNA transcripts following reverse transcription PCR, determining relative or absolute copy number can be difficult due to sample-to-sample variation. The use of a recombinant mRNA internal standard that contains target mRNA primer sequences greatly improves reproducibility of quantitation. Reverse transcription PCR products generated from the internal standard can be distinguished from the product generated from the target gene mRNA because of their size differences. In this report we present a facile and general PCR-based method for synthesis of internal standards that may be used as competitive or co-amplified templates for quantitative reverse transcription PCR.