Real-time polymerase chain reaction quantification of gene expression levels of murine endothelin-A and endothelin-B receptors: gene expression profiles by the standard curve method.
A rapid analysis method for murine endothelin-A (ETA) and endothelin-B (ETB) receptor gene expression levels was established using real-time quantitative reverse transcriptase-polymerase chain reaction. We designed primer pairs and TaqMan probes specific for the two cDNAs and available for mouse and rat systems. The standard curve method was used to examine relative expression. The gene expression levels of ETA and ETB were estimated as gene expression rates by normalizing to the expression of the housekeeping 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 and found that a greater than 1.6-fold increase in relative gene expression is detectable as a significant change. ETA and ETB receptor gene expression was found in all 16 organs of mouse and rat examined, and high levels of expression were observed in the lung, uterus, ovary, intestine, and cerebellum. The gene expression patterns essentially agreed with those determined by RNase protection assay, Northern blot, and conventional endpoint polymerase chain reaction. These results show that this new rapid, sensitive, and semi-automated method is accurate, quantitative, and reproducible. This method is also useful for examining regulation of hormone receptor gene expression under physiological conditions in organs.
Adur
Javier
J
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Uchide
Tsuyoshi
T
Takizawa
Satoshi
S
Quan
Jiexia
J
Saida
Kaname
K
D000818:Animals; D001483:Base Sequence; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D044022:Receptor, Endothelin A / Q000235:genetics*; D044023:Receptor, Endothelin B / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B