Rapid quantitation of mRNA species in ethidium bromide-stained gels of competitive RT-PCR products.
A rapid method for quantifying the low-abundant mRNAs of the low density lipoprotein receptor and the 3-hydroxy-3-methylglutaryl coenzyme A reductase by competitive polymerase chain reaction is presented. This approach requires neither special labeling nor blotting procedures. For each analysis, a defined amount of total cellular RNA is co-reverse transcribed and co-amplified with a titration series of in vitro synthesized competitor RNA that carries an internal deletion. The equivalence point, which defines the amount of specific RNA in the sample, can be scored in ethidium bromide-stained agarose or polyacrylamide gels of the reaction products. As an example, responses to pravastatin, a competitive inhibitor of the HMG-CoA reductase, in a human tumor cell line were analyzed with this new technique. As a control, the expression of the unregulated gene, glyceraldehyde-3-phosphate dehydrogenase was measured in parallel using the same methodology. The results obtained were compared with those obtained by conventional Northern blotting.
Gebhardt
A
A
Institute for Pathology, University of Hamburg, Germany.
Peters
A
A
Gerding
D
D
Niendorf
A
A
D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D004996:Ethidium; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D011973:Receptors, LDL / Q000235:genetics; D013194:Staining and Labeling; D013274:Stomach Neoplasms; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011973:Receptors, LDL; D006903:Hydroxymethylglutaryl CoA Reductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase; D004996:Ethidium