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<p>Abstract</p> <p>Background</p> <p>Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between <it>in vivo </it>and <it>in vitro </it>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 <it>in vivo </it>and fresh and frozen-thawed <it>in vitro </it>embryos.</p> <p>Findings</p> <p>The expression stability of 8 candidate reference genes (<it>ACTB</it>, <it>GAPDH</it>, <it>H2A/I</it>, <it>HPRT1</it>, <it>RPL32</it>, <it>SDHA</it>, <it>TUBA4A</it>, <it>UBC</it>) was determined in 3 populations of equine blastocysts (fresh <it>in vivo</it>, fresh and frozen-thawed <it>in vitro </it>embryos). Application of geNorm indicated <it>UBC</it>, <it>GAPDH</it>, <it>ACTB </it>and <it>HPRT1 </it>as the most stable genes in the <it>in vivo </it>embryos and <it>UBC</it>, <it>RPL32</it>, <it>GAPDH </it>and <it>ACTB </it>in both <it>in vitro </it>populations. When <it>in vivo </it>and <it>in vitro </it>embryos were combined, <it>UBC</it>, <it>ACTB</it>, <it>RPL32 </it>and <it>GAPDH </it>were found to be the most stable. <it>SDHA </it>and <it>H2A/I </it>appeared to be highly regulated.</p> <p>Conclusions</p> <p>Based on these results, the geometric mean of <it>UBC</it>, <it>ACTB</it>, <it>RPL32 </it>and <it>GAPDH </it>is to be recommended for accurate normalization of quantitative real-time PCR data in equine <it>in vivo </it>and <it>in vitro </it>produced blastocysts.</p>