PACS RT-PCR: A Method for the Generation and Measurement of any Poly(A)-Containing mRNA Not Affected by Contaminating Genomic DNA

The generation and measurement of low-abundance messenger RNA (mRNA) transcripts is possible using techniques such as reverse transcriptas-polymerase chain reaction (RT-PCR) (1 ,2 ). There are a number of variables that must be controlled for if accurate and reproducible results are to be obtained (1 ). A significant limitation to the accurate quantitation of many mRNA species is genomic DNA contamination during the RNA purification step (3 ). This contaminating genomic DNA often results in artifactual genomic-derived DNA-PCR amplification products. In most cases, exhaustive DNase digestion of the purified RNA sample is critical, especially in those where exon-specific primers cannot be separated by intronic sequences (3 ). Although DNase digestion appears to work well for poly(A⁺ ) mRNA preparations, DNase pretreatment fails to remove all trace contaminating genomic DNA from total RNA preparations (3 ). In this method, we will demonstrate a quantitative method for the measurement of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA, which we have termed poly(A) cDNA-specific RT-PCR (PACS).