A synthetic standard for use in quantitation of mRNA molecules of GABA(A) receptor subunits: Competitive RT-PCR and its application

Abstract

GABA A receptors are composed of subunits belonging to different families (alpha, beta, gamma, delta, pi, epsilon, and rho) according to degree of sequence homology. At present, the subunit composition of native GABA A receptors in the central nervous system is not known and there are relatively few reports of GABA A receptors in peripheral tissues, where the subunit composition may be simpler and easier to determine. One such tissue is the superior cervical sympathetic ganglion (SCG). GABA and GABA receptors also may play an important role in the early development of the central nervous system. Most of the previous developmental studies on patterns of GABA A receptor subunit expression during development were qualitative reports and some of the quantitative data were incomplete. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method as usually applied is only semiquantitative and has no internal control during first-strand synthesis. In my dissertation project, a simple and sensitive competitive RT-PCR (cRT-PCR) assay was established by synthesizing an artificial internal standard specific for quantification of multiple GABA A receptor subunit mRNAs in both mice and rats. Specifically, by using this technique, 13 known mammalian GABA A receptor subunits were studied in rat superior cervical ganglion (SCG) both qualitatively and quantitatively. Furthermore, the expression of mRNAs encoding alpha4 and alpha5 GABA A receptor subunits during prenatal and postnatal stages were quantitatively analyzed. Also, a regional study and strain comparison for those two subunits were performed. The results have shown that GABA A receptor alpha1-5, beta1-3, gamma1-3 and 6 subunits exist in rat SCG. Quantitative analysis revealed that gamma2, gamma3, beta3, and alpha1 are relatively abundant, although some of the other GABA A receptor mRNAs are also present in very low level. The developmental study showed that alpha4 and alpha5 have different temporal expression profiles. The expression of alpha5 showed an early prominent peak at 3 days postnatally compared to 14 days for alpha4. In the regional study, the hippocampus and basal forebrain showed the highest levels of both alpha4 and alpha5 gene expression. We have demonstrated by using our synthetic internal standard that competitive RT-PCR can provide quantitative information about mRNA levels for many individual GABA A receptor subunits.