Abstract
Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.
Highlights
Bovine cattle have a biological and economic importance in which reproductive traits are a relevant component of livestock and genetic breeding (Cammack et al, 2009)
In order to define the best reference genes (RGs) for bovine in vitro matured oocytes and cumulus cells, eight genes were selected for Quantitative real-time PCR (qPCR) analysis according to their frequency in the previous literature: ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP
In this study the stability of ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP as reference genes was analyzed in order to select the more adequate one to be used in gene expression studies of bovine oocytes and cumulus cells derived from in vitro maturation
Summary
Bovine cattle have a biological and economic importance in which reproductive traits are a relevant component of livestock and genetic breeding (Cammack et al, 2009). Many mRNAs and proteins are synthetized, which regulate oocyte growth and maintain early embryonic development until the embryonic genome is activated (Gosden 2002; Sutton et al, 2003). In this period, the bi-directional interaction between oocyte cumulus and granulosa cells, called crosstalk, is essential to complete oocyte development (Senbon et al, 2003). As a result of this crosstalk, cumulus cells may reveal information about oocyte health through gene expression and many studies investigate these substances as noninvasive molecular markers for good quality oocyte selection (Sugiura et al, 2005; Gilchrist et al, 2008; Hamel et al, 2008)
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