Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts

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 int...

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Bibliographic Details
Published in:BMC research notes 2009-12, Vol.2 (1), p.246-246
Main Authors: Smits, Katrien, Goossens, Karen, Van Soom, Ann, Govaere, Jan, Hoogewijs, Maarten, Vanhaesebrouck, Emilie, Galli, Cesare, Colleoni, Silvia, Vandesompele, Jo, Peelman, Luc
Format: Article
Language:English
Subjects:
Blastocyst
Embryos
Genes
Genetic aspects
Identification and classification
In vitro fertilization
Methods
Physiological aspects
Polymerase chain reaction
Pregnancy
Short Report
Sperm
Veterinary medicine
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Summary: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. 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. 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.
ISSN:1756-0500
1756-0500
DOI:10.1186/1756-0500-2-246