APPLICATION OF QUANTITATIVE RT-PCR TO THE ANALYSIS OF DOPAMINE-RECEPTOR MESSENGER-RNA LEVELS IN RAT STRIATUM

A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR ) procedure has been developed which selectively amplifies and quantif ies the two isoforms of the dopamine D2 receptor. Variability is corre cted by the inclusion of a D2 dopamine receptor mRNA standard within e ach reaction. The internal standard was generated by introducing a poi nt mutation within a D2 cDNA clone that created a unique restriction s ite within the amplified region. An in vitro transcribed RNA for the i nternal mutant control is added to the RNA isolated from brain tissue and the mixture is subjected to RT-PCR, digestion with the restriction enzyme, separation of the products by PAGE, and quantification by dir ect analysis of radioactivity incorporated during the PCR step. The st andard is amplified, in the same reaction as the experimental RNA, usi ng the same primers and RT-PCR conditions. In this manner, the effects of contaminants of the RNA preparation which could affect the amplifi cation procedure are assessed. To insure that the amplification is lin ear, the number of PCR cycles is minimized. This adaptation avoids 'co mpetitive PCR' and provides for a linear response. Moreover, to obviat e non-specific co-amplification, primer annealing steps are performed at or above the melting temperature for the primers, thus increasing s ignal-to-noise ratios. Finally, primer pairs have been designed which permit amplification of specific fragments for each of the five rat do pamine receptor subtypes. These fragments have unique sizes and so can be differentiated when simultaneously amplified in the same RNA prepa rations.