Technical note: Identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots

Abstract

Selection of stable reference genes (REF) is important in real-time PCR data normalization. Bovine tissues such as the mammary gland, liver, muscle, and s.c. fat from the tail head have been thoroughly explored for stable REF, whereas fewer reports exist for other fat depots. Therefore, a suitable combination of REF was tested for different tissues of dairy cattle. Holstein dairy heifers (n = 25) were supplemented (100 g/d) with a control fat (n = 15) without conjugated linoleic acids or with rumen-protected conjugated linoleic acids (n = 10) from the day of calving until slaughter at 1, 42, or 105 d postpartum (n = 5, 10, and 10, respectively). Samples from 6 fat depots (omental, mesenterial, retroperitoneal, s.c. tail head, s.c. withers, and s.c. sternum), liver, semitendinosus muscle, and mammary gland were collected. The REF mRNA were quantified and their stability was analyzed using geNorm(plus). The 3 most stable REF in individual fat tissues and muscle were EMD (emerin), POLR2A (RNA polymerase II), and LRP10 (lipoprotein receptor-related protein 10); in mammary gland were MARVELD1 (marvel domain containing 1), EMD, and LRP10; and in liver were HPCAL1 (hippocalcin-like 1), LRP10, and EIF3K (Eukaryotic translation initiation factor 3). The 3 most stable REF in s.c. fat were EMD, LRP10, and EIF3K; in visceral fat were POLR2A, LRP10, and MARVELD1; and for all 6 adipose tissues were LRP10, EIF3K, and MARVELD1. When the mammary gland was added to the 6 adipose depots, at least 5 REF (LRP10, POLR2A, EIF3K, MARVELD1, and HPCAL1) were needed to reach the threshold of 0.15. Addition of liver to the above-mentioned tissues increased the V value. The data improve the comparison of gene expression between different fat depots. In each case, GAPDH had the lowest stability value.

The influence of maternal energy status during mid-gestation on beef offspring tenderness, muscle characteristics, and gene expression

“…In SUBQ, lipoprotein receptor-related protein 10 (LRP10), RNA polymerase 11 polypeptide A (POLR2A), eukaryotic translation initiation factor 3, subunit K (EIF3K), and emerin (EMD) were used as housekeeping genes, as these genes have been identified as suitable housekeeping genes in bovine fat depots (Saremi, Sauerwein, Danicke, & Mielenz, 2012). Eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and hydroxymethylbilane synthase (HMBS) were utilized as housekeeping genes for both LL and ST samples; these genes were shown to be suitable housekeeping genes for bovine muscular tissue (Perez, Tupac-Yupanqui, & Dunner, 2008).…”

Reference gene selection for quantitative PCR studies in bovine neutrophils

“…In ovine whole blood, SDHA and YWHAZ were considered the most suitable reference genes as they were stably expressed regardless of disease status [12]. In the bovine, potential reference genes have been evaluated in polymorphonuclear leukocytes [14], mammary epithelial cells [15], adipose tissue [16], milk somatic cells [17], and WB-cells [18] of lactating dairy cows, and in muscular tissue [19]; however, to our knowledge, potential reference genes have not been evaluated in WB-neutrophils under conditions of Se supplementation in growing beef calves.…”

Hepatic metabolic response of Holstein cows in early and mid lactation is altered by nutrient supply and lipopolysaccharide in vitro

“…Specific details for total RNA isolation and quality evaluation, cDNA synthesis, primer selection and efficiency evaluation, and quantitative reverse-transcription PCR (qPCR) Kadegowda et al, 2009;Saremi et al, 2012). These 2 reference genes were selected based on their lower M value (measure of reference gene stability; lowest value, greater stability) as recommended by the genorm analysis (M < 0.50) using the Biogazelle q + software (Biogazelle NV, Zwijnaarde, Belgium) and based on the work of Vandesompele et al (2002).…”