Comparative analysis of multiple receptor subunit mRNA in micropunches obtained from different brain regions using a competitive RT-PCR method

Current methods for the quantification of mRNA either require amounts of ti ssue which make the analyses unsuitable for studies of changes in discrete brain nuclei (RNA protection assay) or limit analysis to a few species at a time (in situ hybridization). A method is described which permits comparat ive analysis of the expression of mRNAs obtained from punches of the centra l nervous system. This approach uses a synthetic, PCR-generated, internal s tandard which is directed towards the same sequence as the target message b ut is smaller in length. The internal standard is added to the PCR mix and amplified at the same rate as the target message. The mass of both the endo genous target message and internal standard is then measured, using densito metry, and expressed as the mass ratio of target message to internal standa rd. This approach has been used to compare the expression of mRNA for three receptor subunits: GABA(A-alpha 1), NMDA-R1 and Glu-R1, in two distinct br ain regions: the hypothalamic paraventricular nucleus and the caudal nucleu s of the solitary tract. The mass ratios of target to internal standard fro m the PVN and caudal NTS punches from six rats were measured and averaged. In the PVN, the mass ratio of GABA(A-alpha 1) message (19.80 +/- 1.20, mean +/- SE) was higher than that of NMDA-R1 (0.74 +/- 0.19, p < 0.01) or Glu-R 1 (1.04 +/- 0.07, p < 0.01). Similarly, in the NTS the mass ratio for GABA( A-alpha 1) (10.03 +/- 1.67) was higher than that of NMDA-RI (1.21 +/- 0.13, p < 0.05) or Glu-R1 (0.70 +/- 0.09, p < 0.05). Within both the PVN and NTS , there was no difference between the mass ratios for NMDA-RI and Glu-R1. C omparing the PVN to the NTS, the mass ratios for GABG(A-alpha 1) and Glu-R1 were higher in PVN compared to NTS (p < 0.05), while the ratio for NMDA-R1 was comparable in the two nuclei. The method described permits comparison of relative levels of mRNA expression in anatomically distinct brain nuclei and is appropriate for measuring changes in the receptor subunit compositi on of a given species following interventions or changes in physiological s tate. (C) 1998 Elsevier Science B.V. All rights reserved.