A RAPID AND SENSITIVE PROTOCOL FOR COMPETITIVE REVERSE-TRANSCRIPTASE (CRT) PCR ANALYSIS OF CELLULAR GENES

The specific analysis of gene transcripts is of increasing importance for studies in molecular pathology. Competitive RT-PCR with mutagenize d exogenous competitor templates has evolved as an attractive approach to quantify individual mRNA levels. The generation of exogenous compe titor RNAs usually requires mutagenesis and cloning of the mutant frag ment 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 labori ous cloning of mutant exogenous competitors. Templates for the in vitr o transcription are generated in a single PCR reaction with simultaneo us addition of a promoter sequence 5` of the forward primer and deleti on 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 f or in vitro transcription to yield exogenous competitor RNA of equal q uality and amount as conventional cloning strategies. Total RNA amount s are corrected for by analyzing the expression of different housekeep ing 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 semiautomat ed sequencer. In the present study, this protocol was employed to anal yze the expression of the PTCH, Fas-receptor, NF-1, beta S-microglobul in and GAPD genes in human brain tumors. It will, however, be widely a pplicable to studies on cellular transcripts in biological specimens.