Measurement of mRNA by quantitative PCR with a nonradioactive label

Powell, Elizabeth E. and Kroon, Peter A. (1992) Measurement of mRNA by quantitative PCR with a nonradioactive label. Journal of Lipid Research, 33 4: 609-614.

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Author Powell, Elizabeth E.
Kroon, Peter A.
Title Measurement of mRNA by quantitative PCR with a nonradioactive label
Journal name Journal of Lipid Research   Check publisher's open access policy
ISSN 0022-2275
1539-7262
Publication date 1992-04-01
Year available 1992
Sub-type Article (original research)
Open Access Status File (Publisher version)
Volume 33
Issue 4
Start page 609
End page 614
Total pages 6
Place of publication Rockville, MD, United States
Publisher American Society for Biochemistry and Molecular Biology,
Language eng
Abstract This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.
Keyword Gene Expression
Regulation
Cholesterol Metabolism
Digoxigenin-11-Dutp
3-Hydroxy-3-Methylglutaryl Coenzyme-A Reductase
Low Density Lipoprotein Receptor
Chemiluminescence
DNA
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: UQ Centre for Clinical Research Publications
School of Biomedical Sciences Publications
School of Medicine Publications
 
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