A sensitive RNase protection assay for the quantitation of the mRNAs for the LDL receptor and HMG-CoA reductase in human total RNA. Effects of treatments on cells in culture designed to up- and down-regulate expression of the LDL receptor.
Regulation of expression of the genes for the low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is of central importance in the control of cholesterol metabolism and thus in influencing the concentration of low density lipoprotein in the plasma. This can be studied by investigating the effects of factors (hormones, drugs, etc.) on the levels of mRNA for these genes. An RNase protection assay is reported for measurement of the levels of mRNA for the LDLR and HMGR. Several probes have been developed for these genes, together with probes for the "housekeeping" genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Various conditions in the assay have been examined and optimised, e.g. conditions for solution hybridization and RNase digestion and the use of "sense" RNA standards. The assay allows accurate measurement of approximately 2 x 10(7) copies of LDLR and HMGR mRNAs, which is equivalent to the number of copies present in approximately 1 x 10(6) human dermal fibroblasts and approximately 5 x 10(5) Hep G2 liver hepatoma cells cultured in 10% fetal calf serum. The average number of copies of mRNA per cell was estimated in fibroblasts and Hep G2 cells under various conditions of regulation of the LDLR and revealed the following: [table: see text] Under the chosen conditions 10 copies per cell was the detection limit for the assay. The effect of these treatments on the number of copies of mRNA per cell for beta-actin and glyceraldehyde-3-phosphate dehydrogenase was also determined.
Le Cras
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University of Cambridge, Department of Pathology, U.K.
Gherardi
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Bowyer
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D000199:Actins / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts; D005786:Gene Expression Regulation; D005986:Glyceraldehyde 3-Phosphate / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics*; D008114:Liver Neoplasms, Experimental; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D011973:Receptors, LDL / Q000235:genetics*
D000199:Actins; D015347:RNA Probes; D012333:RNA, Messenger; D011973:Receptors, LDL; D005986:Glyceraldehyde 3-Phosphate; D006903:Hydroxymethylglutaryl CoA Reductases