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.
Highlights
A major difficulty in studying the regulation of HMGCoA reductase and low density lipoproteins (LDL)-R gene expression in humans in vivo has been the limited amount of tissue available for RNA analysis
We de'scribe the application of this method to the measurement of LDL receptor (LDL-R) and HMGCoA reductase mRNA levels in circulating peripheral blood mononuclear (PBMN) cells and human liver biopsy samples
Since the same primers are used in the polymerase chain reaction (PCR) amplification of both templates, there are no differences in primer efficiency for cDNA derived from cRNA or mRNA
Summary
Summary 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. 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 3hydroxy-3methylglutaryl (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-3methylglutarylcoenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed.
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