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Alexander Beck, Rainer Lehmann, Giovanni Gambaro, Hans-Ulrich Häring, Erwin D. Schleicher, Wolfgang Voelter, Monica Ceol
Advances in Reverse Transcription Polymerase Chain Reaction Analysis of Cellular mRNA Levels of Transforming Growth Factor-?1 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection
The prosclerotic transforming growth factor ?1 (TGF-?1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-?1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is timeconsuming (? 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 37, 05/1999
Pages: 527 - 532