Quantitation of human brain GABA A receptor β isoforms by competitive RT–PCR

Abstract

We have developed a competitive RT–PCR assay, adapted from Lewohl et al. [Brain Res. Brain Res. Protoc. 1 (1997) 347], for the quantitation of GABA A receptor β isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the β 1 sequence except for a 61 bp deletion and the incorporation of a Hind III restriction enzyme site. Unlike traditional competitive RT–PCR, which requires a range of internal standard concentrations to be titrated against a constant amount of unknown, this method relies on a standard curve for quantitation of each sample and thus permits increased sample throughput. This method is suitable for the quantitation of β 1, β 2 and β 3 isoforms of the GABA A receptor in human alcoholic and control brain.