Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle

Abstract

Achieving greater understanding of the genomic influence on milk synthesis in dairy cows represents a daunting challenge. Bovine-specific microarrays have allowed for high-throughput gene expression analysis of the mammary transcriptome. However, real-time PCR (qPCR) still represents the method of choice for accurate expression profiling of small numbers of genes and verification of key microarray relationships. This method is extremely sensitive but requires data normalization to account for analytical errors. Ideally, expression of genes used as internal controls should not be affected by specific treatments or physiological state. Mammary biopsies were collected from five cows each at -15, 1, 15, 30, 60, 120, and 240 days relative to parturition for gene expression profiling. We evaluated expression of nine genes (RPS9, ACTB, GAPD, GTP, ITGB4BP, MRPL39, RPS23, RPS15, and UXT) that could serve as internal controls in mammary tissue using qPCR. Due to gradual increases in mammary RNA concentration (mug/mg tissue) over lactation, all genes investigated experienced a dilution effect. We used pairwise comparison of expression ratios to analyze the reliability of these genes as internal controls. UXT, RPS9, and RPS15 had the most stable expression ratios across cow and time. We also assessed co-regulation among genes through network analysis. Network analysis suggested co-regulation among most of the genes examined, with MYC playing a central role. Pairwise comparison was suitable for finding appropriate internal controls in mammary gland tissue. Results showed that the geometrical average of UXT, RPS9, and RPS15 expression could be used as internal control for longitudinal mammary gene expression profiling.

Gene expression of six major milk proteins in primary bovine mammary epithelial cells isolated from milk during the first twenty weeks of lactation

“…We hypothesized that milk protein gene expression has a pivotal effect on milk protein composition whereas milk protein concentration was not influenced. This assumption is confirmed by Bionaz and Loor (2007).…”

Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells

“…As a routine, 0.15 was proposed as the cut-off value for V value (Vandesompele et al, 2002), below which the inclusion of an additional control gene is not required. Sometimes, a strict cut-off of 0.10 was used (Bionaz and Loor, 2007). In our study, all of the V n/n + 1 were less than 0.10 (Figure 3), indicating increasing number of genes does not significantly change the value of the normalization analyses.…”

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

“…RPS15 had been demonstrated to be a RG in humans (Kitagawa et al., 1991; Shiga, Yamamoto, & Okamoto, 1990) and is a suitable RG in many cases in mammals (Bionaz & Loor, 2007; Kumar et al., 2012). Interestingly, the least stable genes according to our stability ranking included some genes widely used as RGs in previous studies (e.g., ACT and 18S ).…”