QUANTITATION OF DOPAMINE D-2 RECEPTOR MESSENGER-RNA IN A MESENCEPHALIC CELL-CULTURE USING A NONRADIOACTIVE COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION METHOD

Studies on gene expression during differentiation and maturation proce sses have to cope with determinations of extremely low steady state le vels of specific mRNA. Using the experimental model of dopamine D-2 re ceptor (D2R) expression in a primary mesencephalic cell culture we wor ked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells pres ent in a few wells of the culture. The method uses an internal cRNA st andard which shares both primer binding sites and PCR product length w ith the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the di stinction of internal standard and target sequences followed by the ch emiluminescent detection of hybridized DNA. Applying this method the l evels of D-2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/mu g RNA and increased to about 1200 fg/mu g RNA on day in vitro 13-15. (C) 1998 Published by Elsevier Sci ence B.V. All rights reserved.