title: Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts.
abstract: BACKGROUND
  Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos.
  
  
  RESULTS
  The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated.
  
  
  CONCLUSIONS
  Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.
authors.affiliation: Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. karen.goossens@ugent.be.
authors.forename: Katrien
authors.initials: K
authors.lastname: Smits
authors.forename: Karen
authors.initials: K
authors.lastname: Goossens
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
authors.forename: Jan
authors.initials: J
authors.lastname: Govaere
authors.forename: Maarten
authors.initials: M
authors.lastname: Hoogewijs
authors.forename: Emilie
authors.initials: E
authors.lastname: Vanhaesebrouck
authors.forename: Cesare
authors.initials: C
authors.lastname: Galli
authors.forename: Silvia
authors.initials: S
authors.lastname: Colleoni
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.forename: Luc
authors.initials: L
authors.lastname: Peelman