engleski

Semi-quantitative RT-PCR: development of the method for characterization of monoamine oxidase A and B gene expression in rat brain

Reverse transcription-polymerase chain reaction (RT-PCR) is a sensitive and powerful technique for mRNA detection. However, its quantitation seems to be more complex. Namely, absolute mRNA quantitation requires either laborious internal standard construction (competitive RT-PCR) or expensive equipment (Real-time RT-PCR). On the other hand, there is a possibility of relative quantitation (semi-quantitative RT-PCR) of different mRNA species by the use of various house-keeping genes (e.g.  -actin, glyceraldehyde-3-phosphate dehydrogenase, cyclophylin etc.) as referent measures. In this study, development of a method for relative quantitation of genes encoding monoamine oxidase A and B (MAO-A and MAO-B) in the rat brain is presented. Total RNA was isolated from the two brain regions (frontal cortex and raphé nuclei). cDNA was synthesized by the use of Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo dT primers. PCR was then performed using specific oligonucleotide primers for  -actin, cyclophylin B, MAO-A and MAO-B. The range of linear amplification was investigated in each region by varying number of PCR cycles from 18 to 42 (range was chosen for each gene specifically). PCR products were subjected to electrophoresis on 1, 5% agarose gel and visualised by 0, 1% ethidium bromide staining. Gels were scanned and digital images subjected to densitometric analysis using appropriate software. The results of this study enabled us to set all experimental parameters required for reliable estimation of levels of expression of MAO-A and B genes in the rat brain. This metodology was specifically developed for future studies of the MAO-A and B gene expression in rat sublines with altered serotonin homeostasis originally developed in our laboratory by selective breeding procedure.

semi-quantitative RT-PCR; expression; monoamine oxidase; rat brain

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