Advances in reverse transcription polymerase chain reaction analysis of cellular mRNA levels of transforming growth factor-beta1 by capillary electrophoresis with laser-induced fluorescence detection.
The prosclerotic transforming growth factor beta1 (TGF-beta1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-beta1 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 time-consuming (> or = 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.
Abteilung für Innere Medizin IV, Indokrinologie, Stoffwechsel und Pathobiochemie, Medizinische Klinik und Poliklinik der Universität, Tübingen, Germany.
A
A
Beck
R
R
Lehmann
G
G
Gambaro
H U
HU
Häring
E D
ED
Schleicher
W
W
Voelter
M
M
Ceol
D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005920:Glomerular Mesangium / Q000737:chemistry; D006801:Humans; D007834:Lasers; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D016212:Transforming Growth Factor beta / Q000235:genetics*
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta