HOW TO CORRECT FOR ERRORS IN MESSENGER-RNA QUANTITATION BY COMPETITIVE PCR DUE TO HETERODUPLEX FORMATION OF AMPLIFICATION PRODUCTS

The Q-RT-PCR method for determining mRNA levels is an internally-contr olled quantitative reverse transcriptase linked polymerase chain react ion to assess gene activity by monitoring the accumulated stable trans cript level, in a given sample of extracted total RNA. Internal and co mpetitive standards are used for mRNA titration because of the exponen tial nature of PCR; possible variations in RT efficiency are corrected for by the use of riboprobe RNA standards. A renin mRNA assay has bee n optimized by altering PGR primer parameters. Q-RT-PCR suffers from t he occurrence of heteroduplex DNA formation between amplification prod ucts from homologous standard and target mRNA. Co-migration of various PCR products is shown to occur on neutral agarose gels, and this will lead to serious errors in mRNA determinations. Adjustment of the stan dard electrophoresis conditions is required for absolute mRNA quantita tion by Q-RT-PCR.