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.