MODIFIED METHOD FOR COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION FOR RAPID AND AUTOMATED QUANTITATION OF MESSENGER-RNA IN MULTIPLE SAMPLES

Background: Competitive reverse transcription polymerase chain reactio n (RT-PCR) has been used increasingly to quantitate messenger RNA (mRN A) levels; however, conventional competitive RT-PCR methods require fo ur or five reactions per sample of RNA, employing serial dilutions of an internal competitor sequence, making analysis of multiple samples a tedious process. A modified method is described by which multiple sam ples and multiple RNA transcripts can be analyzed easily by an automat ed process. Methods and Results: Transforming growth factor beta-1 (TG F-beta-1) mRNA was assayed in total RNA extracted from cultured human skin fibroblasts. A standard solution of total RNA was first prepared by pooling RNA from several cell lines and stored in aliquots. A 270-b p competitor RNA molecule (RNA mimic) was prepared by in vitro transcr iption and was added to each reaction, PCR was performed with a fluore scent dye (Hex; Applied Biosystems, Foster City, CA)-labeled sense pri mer to amplify a 161-bp-long c DNA product of target TGF-beta-1 mRNA s equence and the RNA mimic. The PCR products were analyzed with an auto mated laser-scanned gel electrophoresis system and the area under the curve (AUG) was used for quantitation. The concentration of TGF-beta-1 mRNA in standard RNA was determined by conventional competitive RT-PC R. Subsequently, equal amounts of RNA mimic were mixed with four seria l dilutions of standard RNA and 0.1 mu g of sample total RNA for RT-PC R. A standard curve was generated using the known dilutions of a stand ard target RNA solution and ratio of AUC for target to that for mimic for each dilution. The unknown sample was then quantitated by interpol ation of its area under the curve ratio on the standard curve. This me thod had inter-and intra-assay coefficients of variation of less than 10%. Conclusions: This modification is highly reproducible for quantit ation of mRNA and significantly reduces the number of PCR reactions re quired for each assay. It can be used to assay several RNA molecules i n a given sample by designing RNA mimics and PCR primers to generate P CR products of different lengths so that they can be analyzed by the l aser scanning of a single lane of electrophoretic gel.