QUANTIFICATION OF G-PROTEIN MESSENGER-RNA USING REVERSE TRANSCRIPTIONAND COMPETITIVE PCR WITH A COLORIMETRIC MICROPLATE ASSAY

This paper reports an assay for the quantification of levels of specif ic mRNA for the alpha subunits of the inhibitory G proteins G(alpha i- 1), G(alpha i-2), and G(alpha i-3). The assay employs reverse transcri ption and competitive polymerase chain reaction (PCR) coupled to enzym e-linked oligonucleotide sorbent assay for differential detection and quantification of PCR products. The assay was conducted with conventio nal thermal block PCR cyclers as well as rapid air microcapillary cycl ers. The detection stage consists of three steps using synthetic oligo nucleotides, commercially available reagents and a conventional 96-wel l plate absorbance reader at settings of 450 and 630 nm. The assay is: (1) rapid, requiring about 3 h For quantification of PCR products; (2 ) safe, being non-radiometric; (3) relatively simple; (4) highly sensi tive, being capable of detecting less than 10 initial copies of target cDNA; (5) precise, resolving two-fold differences in initial copy num bers of specific sequences as low as 10(-20) mel; (6) linear over a 3 log range, with two-fold differences in the quantity of cDNA producing consistent reductions in quantity of specific cDNA detected; and (7) reproducible, intra-assay and inter-assay coefficients of variation be ing 11.9 and 14.7%, respectively. (C) 1998 Academic Press Limited.