Effect of RNA concentration on cDNA synthesis for DNA amplification.

1The Rheumatology Section, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; 2The Pediatric Rheumatology Section, The Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; 3The Biotechnology Center, The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 DNA amplification utilizing the polymerase chain reaction (PCR) has greatly enhanced the ability to detect the presence of rare mRNA species in cells. By employing specific sense and antisense oligonucleotide primers with thermostable DNA polymerase, PCR allows amplification of target sequences by several log orders of magnitude. (l'z) This technique is being used increasingly to quantify rare mRNA species from cells. (3-6) In these studies, it is critical that the initial cDNA synthesis step be well controlled and optimized to produce a uniform substrate for amplification. We have examined RNA concentration in the initial cDNA synthesis reaction to optimize it for PCR amplification of both highand low-abundance messages.