Quantitation of the sterol regulatory element-binding protein mRNA in mononuclear blood cells by competitive RT-PCR.
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.
D000214:Acyl Coenzyme A / Q000096:biosynthesis / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D016014:Linear Models; D008055:Lipids / Q000097:blood; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D011973:Receptors, LDL / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D000214:Acyl Coenzyme A; D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008055:Lipids; D012333:RNA, Messenger; D011973:Receptors, LDL; C497387:SREBF1 protein, human; C497390:SREBF2 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases