Determination of mRNA levels of cholesterol biosynthesis enzymes and LDL receptor using ribonuclease protection assay.
We designed a rapid method for determining mRNA content of cholesterol biosynthesis enzymes and LDL receptor (LDLR) using a ribonuclease protection assay (RPA). 32P-labeled cRNA fragments for genes of human LDLR and the enzymes HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), farnesyl pyrophosphate synthase (FPPS), and squalene synthase (SQS) were prepared by in vitro transcription. Total RNA prepared from HepG2 cells was hybridized with the cRNA probe and the hybridized mRNA was determined under protection from RNase digestion. Probe content used in this assay was excess in determining the desired mRNA in total RNA, and surplus probes were completely digested using RNase under standard conditions. When cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), mRNA levels of FPPS, SQS, and LDLR were about 4- to 7-fold higher than those of HMGS, HMGR, and MK. On incubation with DMEM supplemented with 10% lipoprotein-deficient serum (LPDS) for 8 h, all messenger RNA levels increased 1.5- to 3.5-fold. In addition, when the HMG-CoA reductase inhibitor compactin was added to 10% LPDS-DMEM, these levels increased even further and the change in mRNA level seemed to differ between the enzymes and LDLR. From these results, we conclude that RPA is a useful method for determining the very small amount of mRNA level of cholesterol biosynthesis enzymes and LDLR in the cell.
Shimokawa
T
T
Third Department of Internal Medicine, University of Tokyo, Japan.
Kawabe
Y
Y
Honda
M
M
Yazaki
Y
Y
Matsumoto
A
A
Itakura
H
H
Kodama
T
T
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002784:Cholesterol / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000737:chemistry; D004122:Dimethylallyltranstransferase / Q000235:genetics; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics; D007700:Kinetics; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D011973:Receptors, LDL / Q000235:genetics*; D012260:Ribonucleases; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D015347:RNA Probes; D012333:RNA, Messenger; D011973:Receptors, LDL; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D006904:Hydroxymethylglutaryl-CoA Synthase; D004122:Dimethylallyltranstransferase; D013186:Farnesyl-Diphosphate Farnesyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D012260:Ribonucleases