Reference Gene Selection for Gene Expression Analysis of Oocytes Collected from Dairy Cattle and Buffaloes during Winter and Summer

Abstract

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.

Developmental competence and expression profile of genes in buffalo (Bubalus bubalis) oocytes and embryos collected under different environmental stress

“…The candidate reference genes (YWHAX and GAPDH) were chosen based on a previous study using buffalo's oocytes during summer and winter (Macabelli et al 2014). The target genes were selected taking into account their roles in developmental competence (OCT-4, IGF-2R and GDF-9), heat stress (HSP-70.1), oxidative stress (MnSOD), glucose metabolism (GLUT-1) and apoptosis (BAX and BCL-2).…”

Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)

“…Therefore, number of studies have successfully been conducted to identify least regulated and stably expressed RGs [9][10][11] across tissues of various species such as human, pig, sheep, bovines etc [12][13][14][15][16][17][18]. To our knowledge, not much information is available on set of suitable reference genes that can be used in lactation or physiological studies in riverine buffaloes (Bubalus bubalis) although few studies have been performed in specific cell types/tissues [19][20][21]. Considering the fact that choice of suitable reference/RGs genes is crucial for accurate expression profiling of target genes, this study presents the information on panel of appropriate RGs across 12 buffalo tissues.…”

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

“…The primers were also sequenced to test their specificity. DNTM1, DNMT3A, DNMT3B and the reference genes PPIA and RPL15 were used as described previously 54,55 . Amplification were performed with initial denaturation at 95°C for 2 minutes, following by 45 cycles at 95°C for 15 seconds and 60°C for 1 minute, followed by melting curve analysis to verify the presence of a single amplified product.…”