Application of the Random Arbitrary Primed Polymerase Chain Reaction Differential Display Method to Isolate Genes of Cholesterol Metabolism-related Proteins from Rat Liver

Abstract

The random arbitrary primed (RAP) polymerase chain reaction (PCR) differential display (DD) method was applied to isolate genes related to cholesterol metabolism from exogenously hypercholesterolemic (ExHC) rats and the progenitor, SD rats. Forty-seven trials of RAP-PCR DD resulted in the isolation of 37 clones differing in strain, cholesterol supplementation or their interaction. Among their fingerprints, five clones gave reproducible patterns by a Northern blotting analysis. The sequence of two clones with lower mRNA abundance in ExHC rats than in SD rats was homologous to that of fatty acid synthase and oxalyl-CoA decarboxylase. Two other clones with higher mRNA on the n-cholesterol diet were matrin F/G protein and the NMDA receptor glutamate-binding subunit. The other clone with higher mRNA abundance in ExHC rats on the cholesterol diet was myelodysplasia/myeloid leukemia factor 2. Fifteen trials of reverse transcriptase (RT)-PCR DD yielded 10 clones, but none of the fingerprints were reproduced by the Northern blotting analysis. These results indicate that RAP-PCR DD is an appropriate alternative to RT-PCR DD for isolating the genes involved in hypercholesterolemia.