Quantitation of human brain GABAA receptor beta isoforms by competitive RT-PCR.
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 beta isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the beta(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 beta(1), beta(2) and beta(3) isoforms of the GABA(A) receptor in human alcoholic and control brain.
Buckley
S Tracey
ST
Neuroscience Unit, Department of Biochemistry, University of Queensland, Brisbane, Australia.
Dodd
Peter R
PR
D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011963:Receptors, GABA-A / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards
D020033:Protein Isoforms; D012333:RNA, Messenger; D011963:Receptors, GABA-A