Validating reference microRNAs for normalizing qRT-PCR data in XXXX oocytes and preimplantation embryos.

@entity69539 @entity71142 @entity69540 @entity71141 @entity56452 @entity71143 @entity291
BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. RESULTS: We analyzed the suitability of a number of miRNAs as potential expression normalizers in @entity291 oocytes and early embryos, and porcine oocytes. The stages examined were @entity291 oocytes at the germinal vesicle (GV) and metaphase II stages, @entity291 zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of @entity71141 , miR-103, miR-26a, @entity69539 , @entity71142 , Let-7a and U6 for @entity291 samples and @entity56452 , miR-26a, @entity71141 , miR-103, @entity69540 , @entity71143 and @entity69539 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified @entity71141 and miR-103 as the most stably expressed in @entity291 samples and miR-26a, @entity69539 and @entity71141 in porcine samples. CONCLUSIONS: The combination of @entity71141 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on @entity291 oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, @entity69539 and @entity71141 .
