Advances in Reverse Transcription Polymerase Chain Reaction Analysis of Cellular mRNA Levels of Transforming Growth Factor-β1 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection
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Alexander Beck
Abstract
The prosclerotic transforming growth factor β1 (TGF-β1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-β1 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.
Copyright (c)1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Metrological Support of the Institute for Reference Materials and Measurements (IRMM) to Clinical Chemistry
- Web Applications for Total Quality Management
- Inflammation, the Acute Phase Response and Atherosclerosis
- Characterization of Two Distinct Mechanisms for Induction of Apoptosis in Human Vascular Endothelial Cells
- Lipid Peroxidation and Antioxidant Defenses in Cystic Fibrosis Patients
- Autoantibodies against Oxidised Low-Density Lipoprotein in Patients with Obstructive Sleep Apnoea
- Inter-α-Inhibitor (IαI) Concentration in Pig Plasma Is Independent of Acute-Phase Protein Response
- Advances in Reverse Transcription Polymerase Chain Reaction Analysis of Cellular mRNA Levels of Transforming Growth Factor-β1 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection
- Determination of Ascorbic Acid in Plasma and Urine by High Performance Liquid Chromatography with Ultraviolet Detection
- A New Liquid Homogeneous Assay for the Determination of HDL-Cholesterol
- On the Standardization of Total Prostate-Specific Antigen: an Exercise with Two Reference Preparations
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- Evaluation of an Automated Method for Cytokine Measurement Using the Immulite® Immunoassay System
- Handbook of Interpretation of Diagnostic Tests by Jacques Wallach
- Assessment of a Major Academic Development Programme Linking European Union with Central and Eastern Europe
