Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.The research was granted by São Paulo Research Foundation (FAPESP; grant numbers 2009/00938-3, 2010/13384-3, 2010/09561-7, 2011/14207-0 and 2012/07510-1), National Counsel of Technological and Scientific Development (CNPq; grant number 476229/2011-1) and Coordination for the Improvement of Higher Level Personnel (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Effect of reference gene selection on expression values of non-reference target genes in immature oocytes from Holstein cattle.

<p>Oocytes were retrieved from heifers (H; n = 17 and 15 oocytes, respectively), high-producing cows in peak lactation (PL; n = 16 and 18 oocytes, respectively), and repeat-breeder cows (RB; n = 17 and 17 oocytes, respectively) during winter (W) and summer (S). The amounts of <i>HSP90AA1</i> (A–E) transcripts are expressed in relation to H during winter because this was considered as a reference group (see Material and methods for more information). Expression values were normalized either by <i>YWHAZ</i> (A), <i>RPL15</i> (B), <i>TPB</i> (C), geometric mean of <i>RPL15</i>, <i>PPIA</i> and <i>GUSB</i> (D) or not normalized (E). P values for animal category, season and animal category*season are denoted in the insets above each graphic. Different letters over bars denote a significant difference among categories within season (P<0.05). *Difference between seasons within category (P<0.05).</p

Effect of season on expression of candidate reference genes in immature oocytes from Holstein cattle.

<p>Oocytes were retrieved from heifers (H; n = 17 and 15 oocytes, respectively), high-producing cows in peak lactation (PL; n = 16 and 18 oocytes, respectively), and repeat-breeder cows (RB; n = 17 and 17 oocytes, respectively) during winter (W) and summer (S). The amounts of <i>ACTB</i> (A), <i>GAPDH</i> (B), <i>GUSB</i> (C), <i>HIST1H2AG</i> (D), <i>HPRT1</i> (E), <i>PPIA</i> (F), <i>RPL15</i> (G), <i>SDHA</i> (H), <i>TBP</i> (I) and <i>YWHAZ</i> (J) transcripts are expressed in relation to H during winter because this was considered as a reference group (see Material and methods for more information). P values for animal category, season and animal category*season are denoted in the insets above each graphic. Different letters over bars denote a significant difference among categories within season (P<0.05). *Difference between seasons within category (P<0.05).</p

Effect of reference gene selection on expression values of non-reference target genes in immature oocytes from buffaloes.

<p>Oocytes were retrieved from nulliparous (N; n = 12 and 11 oocytes, respectively) and multiparous (M; n = 11 and 13 oocytes, respectively) during winter (W) and summer (S). The amounts of <i>HSPA1A</i> plus <i>HSPA1B</i> (termed in the current work as <i>HSPA1AB</i>; A–D) and <i>HSP90AA1</i> (E–H) transcripts are expressed in relation to N during winter because this was considered as a reference group (see Material and methods for more information). Expression values were normalized either by <i>TBP</i> (A and E), <i>YWHAZ</i> (B and F), geometric mean of <i>YWHAZ</i>, <i>GUSB</i> and <i>GAPDH</i> (C and G) or not normalized (D and H). P values for animal category, season and animal category*season are denoted in the insets above each graphic.</p

Effect of season on expression of candidate reference genes in immature oocytes from buffaloes.

<p>Oocytes were retrieved from nulliparous (N; n = 12 and 11 oocytes, respectively) and multiparous (M; n = 11 and 13 oocytes, respectively) during winter (W) and summer (S). The amounts of <i>ACTB</i> (A), <i>GAPDH</i> (B), <i>GUSB</i> (C), <i>HPRT1</i> (D), <i>PPIA</i> (E), <i>RPL15</i> (F), <i>SDHA</i> (G), <i>TBP</i> (H) and <i>YWHAZ</i> (I) transcripts are expressed in relation to N during winter because this was considered as a reference group (see Material and methods for more information). P values for animal category, season and animal category*season are denoted in the insets above each graphic.</p