Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining

We developed a protocol for quantification of relative gene expression usin g reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1), subu nit (GABA(A)R beta(1)) and phospholipase C beta(4), subtype (PLC beta(4),) in rat cerebrum and cerebellum were determined by comparing the ratio of PC R products generated by linear amplification of the target cDNA segments an d glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a refere nce. The density of PCR products was measured from digitized images of phot ographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a singl e band) of the detection system. The accuracy of the present method was <2- fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLC beta(4), was significantly higher in c erebellum than cerebrum. and that of GABA(A)R beta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter rece ptor and intracellular signaling enzyme gene families. (C) 2000 Elsevier Sc ience B.V. All rights reserved.