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 tissue 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 comparative analysis of the expression of mRNAs obtained from punches of the central nervous system. This approach uses a synthetic, PCR-generated, internal standard which is directed towards the same sequence as the target message but 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 endogenous target message and internal standard is then measured, using densitometry, and expressed as the mass ratio of target message to internal standard. This approach has been used to compare the expression of mRNA for three receptor subunits: GABAA-alpha 1, NMDA-R1 and Glu-R1, in two distinct brain regions: the hypothalamic paraventricular nucleus and the caudal nucleus of the solitary tract. The mass ratios of target to internal standard from the PVN and caudal NTS punches from six rats were measured and averaged. In the PVN, the mass ratio of GABAA-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-R1 (1.04 +/- 0.07, p < 0.01). Similarly, in the NTS the mass ratio for GABAA-alpha 1 (10.03 +/- 1.67) was higher than that of NMDA-R1 (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-R1 and Glu-R1. Comparing the PVN to the NTS, the mass ratios for GABAA-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 composition of a given species following interventions or changes in physiological state.
Durgam
V R
VR
Department of Pharmacology, University of Texas Health Science Center, San Antonio 78284-7764, USA. durgam@uthscsa.edu
Mifflin
S W
SW
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D017931:DNA Primers; D017403:In Situ Hybridization / Q000379:methods; D046911:Macromolecular Substances; D008297:Male; D008832:Microchemistry / Q000379:methods; D010286:Paraventricular Hypothalamic Nucleus / Q000378:metabolism; D011677:Punctures; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000235:genetics*; D011963:Receptors, GABA-A / Q000235:genetics*; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017552:Solitary Nucleus / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D046911:Macromolecular Substances; D012333:RNA, Messenger; D018091:Receptors, AMPA; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate