Quantification of GABA(A) receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology.
We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.