COMPETITIVE PCR FOR QUANTITATION OF GONADOTROPIN-RELEASING-HORMONE MESSENGER-RNA LEVEL IN A SINGLE MICROPUNCH OF THE RAT PREOPTIC AREA

A competitive polymerase chain reaction (PCR) for quantitating gonadot ropin-releasing hormone (GnRH) mRNA level in a single micropunch of th e rat preoptic area (POA) is described. The POA (600 mu m in depth) wa s micropunched from frozen rat brain slices and used for mRNA isolatio n using Dynabeads-oligo(dT) magnetic separation technique. The target RNA combined with a synthetic, deletion mutant GnRH cRNA as an interna l standard, is co-reverse transcribed, and their cDNAs are subsequentl y co-amplified by Tag DNA polymerase in the same tube in which the sam e GnRH primers are used. This PCR protocol is sensitive enough to dete ct GnRH mRNA level in a single POA micropunch derived from an individu al rat. There is a linear increase of the amount of GnRH PCR products as a function of input RNA and of the number of PCR cycles. Addition o f mutant GnRH cRNA as an internal standard allows us to quantitate GnR H mRNA level in biological samples and to compensate variations of PCR reaction between samples. Following preoptic treatment with 5'-ADMP; which depletes selectively norepinephrine (NE), GnRH mRNA level was si gnificantly reduced. This simple. vet highly sensitive PCR method appe ars to be a valuable tool for the study of the cellular and molecular regulation of GnRH gene expression in a variety of experimental models .