|
 |
| home > ebr > autumn 2003 > quantification of gene expression by combining competitive rt-pcr with hplc analysis |
|
|
 PUBLICATIONS |
European BioPharmaceutical Review
|
Today, gene expression analysis based on gel electrophoresis of RNA or DNA (after RT-PCR) and different blotting techniques is well-established. Nevertheless, quantification of gene expression also requires densitometric scanning of gels or blots with subsequent calculation of target gene expression levels related to a housekeeping gene or an artificial internal standard. Although a lot of samples could be processed in parallel, the whole process is hampered by many manual operations leading to long analysis times (see Figure 1). Anion exchange HPLC is known to be an effective technique for rapid analysis of double-stranded, single-stranded and plasmid DNA. In principle, these separations are based upon the overall negative charge of DNA molecules, so that smaller species elute prior to larger, more negatively charged molecules.
Competitive RT-PCR is a quantitative adaption of the PCR method in which a known copy number of a (synthetic) RNA or DNA is co-amplified with the target mRNA sample molecule in the same reaction tube. Described is an application of competitive RT-PCR in combination with HPLC-analysis for quantifying gene expression of c-fos coding for the c-fos transcription factor protein, which is under debate to be involved in transcriptional regulation of genes influencing blood pressure regulation.
|
Read full article >>
|
|
 |
|
| Rate this article |
You must be a member of the site to make a vote. |
|
Average rating: |
0 |
| | | | | |
|
|
By Christine Wilde and Georg Schmidt of Tosoh Bioscience; Eva Moellenhoff at the German Institute for High Blood Pressure Research; Bimal Chatterjee at the GSF-National Research Center for Environment and Health, Institute of Development Genetics; and S Nakatani, T Tsuda, Y Yamasaki, H Moriyama, H Watanabe and Y Kato of Tosoh Corporation
Christine Wilde studied Chemistry in Stuttgart and earned the degree Dipl Chemist. She then joined HP, now Agilent, as Application Chemist in Gas Chromatography. She started working for Tosoh Bioscience in 1992 with the development of the quality control department and performance of quality control. Later Christine took over the role of the Technical Support Specialist. Together with Werner Conze she takes care of customer inquiries and problems. She has in-depth knowledge of the product portfolio, especially for the HPLC columns, and assists the realisation of customer seminars and chromatographic workshops.
Dr Georg Schmidt studied Biology at the Technical University Braunschweig and gained his PhD at the Institute of Biotechnology of the research centre Jьlich (Germany). Subsequently, he worked as Postdoc and as Lab Leader at the Biochemie GmbH in Austria. Being involved in variety of projects gave him an in-depth understanding of protein chemistry, enzymatics, immunology, gene expression and validation of analytical methods and downstream processes. The spectrum of methods he applies comprises of mRNA/DNA isolation and blotting techniques including densitometric analysis as well as a wide spectrum of different HPLC and protein purification methods. He joined Tosoh Bioscience GmbH as Technical Manager in 2001.
Eva-Maria Moellenhoff studied Pharmacy at the University of Muenster. She graduated at the University of Heidelberg after studying the effect of the transcription factor expression in the rat brain on the central osmo regulation in the team of Thomas Unger.
The studies were supported by the German Institute for High Blood Pressure Research. She joined GlaxoSmithKline in 1999.
|
|
|
|
|
|
 |
Industry Events |
 |
Inaugural Conference on Pre-Filled Syringes
21-22 January 2009, Crowne Plaza Hotel - St James, London, UK
SMi present their Inaugural Conference on Pre-Filled Syringes 21st and 22nd January 2009 at the Crowne Plaza Hotel - St James, London, UK
More info >> |
|
 |
News and Press Releases |
 |
Catalent Opens New Clinical Supply Warehouse in the UK
14th October 2008
More info >> |
|
 |
