Quantitation of dopamine D2 receptor mRNA in a mesencephalic cell culture using a nonradioactive competitive reverse...

Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA. Using the experimental model of dopamine D2 receptor (D2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture. The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA. Applying this method the levels of D2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/microgram RNA and increased to about 1200 fg/microgram RNA on day in vitro 13-15.