Quantitation of the sterol regulatory element-binding protein mRNA in mononuclear blood cells by competitive RT-PCR

Abstract

Background: The genes for the sterol regulatory element-binding protein-1a (SREBP-1a), -1c, and -2, the low-density lipoprotein (LDL) receptor, and the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play a key role in the intracellular cholesterol and lipid metabolism. Methods: To enable the absolute and relative quantitation of the mRNA levels of these genes we developed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The inclusion of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for reference and normalization enabled us to accurately discriminate between a twofold variance in the expression levels of these genes. We used this assay to study their expression in mononuclear peripheral blood cells (PBMNC). Results: We found that the relative expression of SREBP-1a is tenfold higher than that of SREBP-1c, but only half of that of SREBP-2. The level of SREBP-1a transcripts correlated with that of the SREBP-1c, LDL receptor, HMG-CoA reductase, and SREBP-2 genes, whereas the amount of SREBP-1c mRNA did not show a relationship with that of the latter three genes. The most abundant transcript in PBMNC is that of SREBP-2, followed by that of SREBP-1a, whereas SREBP-1c mRNA is only found in smaller amounts. Conclusions: This competitive RT-PCR method is very well suited for the accurate quantitation of the respective mRNAs.