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BMC Developmental Biology

, 5:27

First Online: 03 December 2005Received: 09 June 2005Accepted: 03 December 2005


BackgroundReal-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used.

ResultsIn this study the transcription levels of 8 commonly used reference genes ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages 2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages.

ConclusionUsing the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-213X-5-27 contains supplementary material, which is available to authorized users.

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Autor: Karen Goossens - Mario Van Poucke - Ann Van Soom - Jo Vandesompele - Alex Van Zeveren - Luc J Peelman


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