The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display |
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Authors: | Norbert Kociok Klaus Unfried Peter Esser Ralf Krott Ulrich Schraermeyer Klaus Heimann |
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Institution: | 1. Department of Vitreoretinal Surgery, University Eye Hospital, University of Cologne, Cologne Joseph-Stelzmann-Strasse 9, D-50931, K?ln, German 2. Department of Toxicology, Medical Institute for Environmental Health at the University of Düsseldorf, Germany 3. Department of Vitreoretinal Surgery, University Eye Hospital, University of Cologne, Cologne, German
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Abstract: | In many applications, an understanding of differentially expressed genes in different tissues, or owing to an applied stimulus
is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification
of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR RAP-PCR] and differential, display DDR-PCR]
has shown, that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR a technique has recently
been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application.
We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing
polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of
differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced
directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived
from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis
completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR)
is a reliable and useful modification of available differential expression methods. |
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Keywords: | Differential gene expression differential display RNA finger-printing semiquantitative RT-PCR heat-stable DNA polymerase with proofreading activity silver stain |
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