Detection of weakly expressed genes in the rostral ventrolateral medulla of the rat using micropunch and reverse transcription-polymerase chain reaction techniques

Abstract

1. The present study describes the use of reverse transcription-polymerase chain reaction (RT-PCR) to detect weakly expressed neurotransmitter receptor mRNA in tissue micropunched from the rostral ventrolateral medulla (RVLM) and other discrete areas of the medulla oblongata of the rat. 2. Micropunches were made from 240 microns transverse medullary sections. Punched regions included the RVLM, hypoglossal nucleus (XIIn), ventrolateral subnucleus of the nucleus tractus solitarius (NTS) and spinal trigeminal nucleus (STN). RNA was extracted and reverse transcribed into cDNA, which was probed for the presence of seven genes: glyceraldehyde phosphate dehydrogenase (GAPDH), neuron-specific enolase (NSE), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), glucocorticoid receptor (GCR), mineralocorticoid receptor (MCR) and the adenosine 5'-triphosphate (ATP) receptor subunit P2X2-1. Each transcript was detected using a semi-nested PCR protocol, which used three primers. 3. Tyrosine hydroxylase was detected in the RVLM and NTS and PNMT was also detected in the RVLM, which agrees with the distribution of catecholamine neurons in the medulla. Expression of GCR mRNA was detected in the RVLM and the XIIn but not in the NTS (it was not probed for in the STN punches). The P2X2-1 receptor message was detected in all areas. Expression of MCR mRNA was detected in the RVLM only. 4. This method offers a simple way to detect the presence of low-abundance receptor mRNA in discrete brain regions.