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

Abstract

Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition. ImplicationsThis study evaluated the expression stability of a set of well-known reference genes in porcine intramuscular stromal-vascular cells (MSVCs), whose adipogenic differentiation is critical for intramuscular fat deposition and pork quality. ACTB, ALDOA and RPS18 were identified to be most stably expressed throughout adipogenic differentiation, and suitable to be used for normalization in quantitative realtime PCR assay, whereas GAPDH was the last choice. This study has provided a valuable data for our further work to explore the molecular mechanism underlying porcine MSVCs adipogenic differentiation and intramuscular fat deposition.

A new set of reference housekeeping genes for the normalization RT-qPCR data from the intestine of piglets during weaning

“…The most frequently used housekeeping genes, GAPDH , B-actin , and 18S [ 9 ], were not identified as ideal endogenous control genes in this study, agreeing with the findings of Nygard et al (2007) [ 34 ], Cinar et al (2012) [ 35 ], Zhang et al (2012) [ 20 ], Gessner et al (2013) [ 36 ], Li et al (2016) [ 37 ], and Wang et al (2016) [ 7 ], but contradicting the findings of Erkens et al (2006) [ 14 ], Kuijk et al (2007) [ 26 ], and Li et al (2011) [ 38 ]. Because RT-qPCR has dynamic range and increased sensitivity compared with traditional quantitation techniques, the well-known housekeeping genes, especially GAPDH , B-actin , and 18S , have been shown to be significantly affected by biological processes, different experimental treatments, and different cell types and tissues [ 39 ].…”

Effects of Intravenous Infusion With Sodium Butyrate on Colonic Microbiota, Intestinal Development- and Mucosal Immune-Related Gene Expression in Normal Growing Pigs

“…One microgram RNA was reverse-transcribed with standard reagents (Biocolors, China). The primers for SCFA uptake [monocarboxylate transporter 1 ( MCT1 )] ( Haenen et al, 2013 ), pro-inflammatory cytokines ( IL-6, IL-8, IL-1β, IL-12p40, IL-18, TNF-α , and IFN-γ ) ( Pié et al, 2004 ; Feng et al, 2014 ; Tudela et al, 2015 ), anti-inflammatory cytokine ( IL-10 and TGF-β ) ( Pieper et al, 2012 ; Feng et al, 2014 ), gene expression regulator [histone deacetylase 1 ( HDAC1 )] ( Lee et al, 2011 ), tight junction protein genes [zonula occludens-1 ( ZO-1 ) and occludin] ( Feng et al, 2014 ), intestine development-related genes [insulin-like growth factor-1 ( IGF-1 ), preproglucagon, IGF-1R , and epidermal growth factor ( EGF ) ( Xiang, 2011 ; Han et al, 2012 ) and housekeeping genes (β-actin and GAPDH ) ( Li et al, 2016 ; Zhang et al, 2017 ) used in this study are presented in Supplementary Table 2 . The target genes and housekeeping genes were measured by quantitative real-time PCR with SYBR Green (Roche, Switzerland) and fluorescence was detected on an ABI 7300 sequence detector.…”

A Key Gene, PLIN1, Can Affect Porcine Intramuscular Fat Content Based on Transcriptome Analysis

“…Intramuscular fat (IMF), corresponding to pork marbling, refers to the fat located within muscles, which is associated with the number and size of intramuscular adipocytes [ 1 ]. Previous studies have indicated that IMF is closely related to flavor, juiciness, and tenderness [ 2 , 3 ]; lower IMF content results in dry and less-flavorful meat, and IMF content is positively associated with sensory quality traits of meat and consumer acceptability [ 4 ]. When IMF content increases from 1 to 3%, sensory quality is significantly improved [ 5 ].…”