Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.
BACKGROUND
Real-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 successful 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.


RESULTS
In 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.


CONCLUSIONS
Using 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.
Goossens
Karen
K
Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. Karen.Goossens@UGent.be
Van Poucke
Mario
M
Van Soom
Ann
A
Vandesompele
Jo
J
Van Zeveren
Alex
A
Peelman
Luc J
LJ
D000818:Animals; D001755:Blastocyst; D002417:Cattle; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D005819:Genetic Markers