title: Evaluation of different strategies for real-time RT-PCR expression analysis of corticotropin-releasing hormone and related proteins in human gestational tissues.
abstract: 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 (DeltaDeltaC (T) or DeltaC (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.
authors.affiliation: Department of Obstetrics and Gynaecology, Endocrinology Laboratory, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Sehringer
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Zahradnik
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Deppert
authors.forename: Michael
authors.initials: M
authors.lastname: Simon
authors.forename: Claudia
authors.initials: C
authors.lastname: Noethling
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Schaefer
mesh_terms: D000465:Algorithms; D002352:Carrier Proteins / Q000032:analysis*; D003346:Corticotropin-Releasing Hormone / Q000032:analysis*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy / Q000378:metabolism*; D018019:Receptors, Corticotropin-Releasing Hormone / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014599:Uterus / Q000378:metabolism*; D000099052:CRF Receptor, Type 1
chemical_list: C101531:CRF receptor type 2; D002352:Carrier Proteins; D018019:Receptors, Corticotropin-Releasing Hormone; C067133:corticotropin releasing factor-binding protein; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone