期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 383, 期 5, 页码 768-775出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-005-0067-9
关键词
real-time PCR; reverse transcription; comparative C-T method; standard curve method; cyclophilin; corticotropin-releasing hormone
In this article real-time quantitative RT-PCR strategies for investigation of mRNA distribution in stress-related corticotropin-releasing hormone (CRH), CRH-binding protein (CRH-BP), and CRH receptors (CRH-R1, CRH-R2) in human gestational tissues are described. The effect of sample and RNA preparation, reverse transcription, and the quantitation strategy were investigated. Both thawing of the sample before homogenization and the RT reaction were identified as critical steps. In contrast, the time-lag from the biopsy until snap-freezing and the homogenization procedure had little effect. The housekeeping gene cyclophilin was found to be differently expressed in gestational tissues, compromising its use as internal reference. For relative quantitation of mRNA levels by TaqMan PCR the standard curve method and the comparative C-T method (Delta Delta C-T or Delta C-T' method) were compared. Both calculation strategies delivered similar relative mRNA amounts in identifying the placenta as the main source of CRH and CRH-BP mRNA compared with myometrium and decidua. Consistent results were also obtained for CRH-R1 and CRH-R2 by both methods of calculation. We conclude that the simpler comparative C-T method is adequate for assessing the mRNA levels of CRH, CRH-BP, and CRH receptors in human gestational tissues.
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