Measurement of mRNA by quantitative PCR with a nonradioactive label.
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.
Powell
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Department of Biochemistry, University of Queensland, Brisbane, Australia.
Kroon
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PA
D001483:Base Sequence; D004247:DNA; D003720:Densitometry; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D008099:Liver / Q000737:chemistry; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000097:blood; D011973:Receptors, LDL / Q000235:genetics
D003856:Deoxyuracil Nucleotides; D012333:RNA, Messenger; D011973:Receptors, LDL; C070202:digoxigenin-11-deoxyuridine triphosphate; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D004076:Digoxigenin