Abstracts

The GABA[sub]A[/sub] receptor plays a critical role in epilepsy. The pentameric chloride channel is formed from seven different classes of subunits with multiple variants, which define its physiological and pharmacological properties. Quantitation of different subunits in various brain regions is important to understand and develop novel drugs for epilepsy. However, conventional RNA assays are tedious and less sensitive. The aim of this study was to develop and validate a novel assay of GABA[sub]A[/sub] receptor subunit gene expression by [quot]real-time[quot] reverse transcriptase and polymerase chain reaction (RT-PCR) and to explore GABA[sub]A[/sub] receptor subunit gene expression in the hippocampus.
Total RNA was extracted from hippocampus and cDNA prepared by reverse transcription. Real-time RT-PCR was applied to quantitate the expression of the target GABA[sub]A[/sub] receptor genes. For each subunit gene, a set of primers and TaqMan[trade] fluorogenic probe was designed, which specifically amplify the target cDNA. TaqMan reaction conditions were optimized and quantitated by the threshold cycle (C[sub]T[/sub], the point during PCR amplification at which fluorescence significantly increases above background) using the iCycler iQ detection system (Bio-Rad). Data was normalized to the housekeeping gene GAPDH.
Real-time TaqMan RT-PCR assay yielded superior sensitivity and accurate expression when compared to traditional RNA assays. The combination of primer mix (600 nM) and TaqMan probe (300 nM) that showed the highest fluorescence was used for all further real-time PCR assays. The method detected very low levels of gene expression (~ 1 pg cDNA). We validated this technology for the quantification of GABA[sub]A[/sub] receptor [alpha]1-4, [beta]2, [gamma]2 and [delta] subunits in the mouse hippocampus. A standard curve for GAPDH and one of the target genes was constructed (3-3000 pg cDNA). The slopes for GAPDH and target genes were around -3.4 (r²=0.998), reflecting similar optimum PCR efficiencies. The ratio of the C[sub]T[/sub] value of the target gene to GAPDH provided the relative expression in the hippocampus. We used this assay to quantify progesterone-induced increase (~ 3-fold) in transcription of GABA[sub]A[/sub] receptor [alpha]1 subunit in the hippocampus.
The study provided a simple and sensitive method for quantification of GABA[sub]A[/sub] receptor subunits in brain tissues. This real-time assay allows rapid, high-throughput transcriptional profiling of the complete GABA[sub]A[/sub] receptor superfamily, and thus provides a new powerful tool for investigations of the mechanisms involved in antiepileptic drugs and GABAergic neurotransmission in seizure susceptibility.
[Supported by: NCSU-CVM]