Rapid quantification of murine endothelin-1 and vasoactive intestinal contractor gene expression levels by a real-time PCR system.
A rapid quantitative analysis method for murine endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC) gene expression levels was established using a real-time polymerase chain reaction (PCR). We designed primer pairs and TaqMan probes specific for murine prepro-ET-1 (PPET-1) and prepro-VIC (PPVIC) genes, based on the cDNA sequence region common to both mouse and rat. The dynamic range for detection in this system spanned 100000-fold of the starting molecule. The gene expression levels of PPET-1 and PPVIC were estimated as gene expression rates normalized by the expression of the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate, which ranged from 16.2 to 55.0% and from 24.2 to 56. 5%, respectively. Using this system, we examined gene expression levels of PPET-1 and PPVIC in mouse tissues. PPET-1 gene expression was found in all tissues at relatively high levels, whereas high levels of PPVIC gene expression were observed only in stomach, intestine, uterus, and ovary. The gene expression patterns agreed well with those determined by RNase protection assay and conventional PCR. These results show that this new rapid method is accurate and reproducible.
Uchide
T
T
Biosignalling Department, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, 305-8566, Ibaraki, Japan.
Adur
J
J
Saida
K
K
D000818:Animals; D017931:DNA Primers; D019332:Endothelin-1 / Q000235:genetics*; D019333:Endothelin-2 / Q000235:genetics; D016232:Endothelins / Q000235:genetics; D005260:Female; D015870:Gene Expression; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010455:Peptides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011498:Protein Precursors / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D019332:Endothelin-1; D019333:Endothelin-2; D016232:Endothelins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011498:Protein Precursors; C059568:vasoactive intestinal constrictor