A Rapid and Sensitive Protocol for Competitive Reverse Transcriptase (cRT) PCR Analysis of Cellular Genes

Abstract

The specific analysis of gene transcripts is of increasing importance for studies in molecular pathology. Competitive RT-PCR with mutagenized exogenous competitor templates has evolved as an attractive approach to quantify individual mRNA levels. The generation of exogenous competitor RNAs usually requires mutagenesis and cloning of the mutant fragment into plasmids followed by in vitro transcription. In contrast to primer directed mutagenesis and in vitro transcription, preparation of the mutant fragments is a time consuming procedure. Here we report on a modified semi-quantitative RT-PCR protocol to circumvent the laborious cloning of mutant exogenous competitors. Templates for the in vitro transcription are generated in a single PCR reaction with simultaneous addition of a promoter sequence 5'of the forward primer and deletion of 10-20 nucleotides at the opposite end just ahead of the reverse primer binding site. The product of this PCR step serves as template for in vitro transcription to yield exogenous competitor RNA of equal quality and amount as conventional cloning strategies. Total RNA amounts are corrected for by analyzing the expression of different housekeeping genes in the same manner. One of the primers used in the following competitive RT-PCR reaction is labeled with a fluorescent dye for the analysis of target and exogenous competitor product on an semiautomated sequencer. In the present study, this protocol was employed to analyze the expression of the PTCH, Fas-receptor, NF-1, beta2-microglobulin and GAPD genes in human brain tumors. It will, however, be widely applicable to studies on cellular transcripts in biological specimens.