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Evaluation of different strategies for real-time RT-PCR expression analysis of corticotropin-releasing hormone and related proteins in human gestational tissues

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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 (ΔΔC T or ΔC 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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Acknowledgements

We gratefully acknowledge the technical assistance of Beate Schmitt and Ruhtraud Ziegler in the development of TaqMan PCR methods. We thank Silke Biller, Ralph Brunner-Spahr, and the staff of the delivery unit for their contributions in collecting the tissue specimens. This work was supported by grant “Scha 467/1–3” from the Deutsche Forschungsgemeinschaft (DFG).

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Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, Endocrinology Laboratory, University of Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany

    Bernd Sehringer, Hans Peter Zahradnik, Wolfgang R. Deppert, Michael Simon, Claudia Noethling & Wolfgang R. Schaefer

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  1. Bernd Sehringer
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  2. Hans Peter Zahradnik
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  3. Wolfgang R. Deppert
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  4. Michael Simon
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  5. Claudia Noethling
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  6. Wolfgang R. Schaefer
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Correspondence to Wolfgang R. Schaefer.

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Sehringer, B., Zahradnik, H.P., Deppert, W.R. et al. Evaluation of different strategies for real-time RT-PCR expression analysis of corticotropin-releasing hormone and related proteins in human gestational tissues. Anal Bioanal Chem 383, 768–775 (2005). https://doi.org/10.1007/s00216-005-0067-9

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  • DOI: https://doi.org/10.1007/s00216-005-0067-9

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