Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR

Feng, Xiaoting; Xiong, Yan; Qian, Hui; Lei, Minggang; Xu, Dequan; Ren, Zhuqing

doi:10.1016/j.jbiotec.2010.09.949

Cited by 46 publications

(33 citation statements)

References 39 publications

(41 reference statements)

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“…In agreement with previous studies (Andersen et al, 2004;Feng et al, 2010;Hurtado del Pozo et al, 2010;Pfaffl et al, 2004), we have observed that the different theoretical methods (NormFinder, geNorm and BestKeeper) provide different outcomes. These discrepancies have been attributed to the different mathematical models used.…”

Section: Discussionsupporting

confidence: 92%

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Selection of reference genes for gene expression studies in rats

Martínez‐Beamonte

Navarro

Larraga

et al. 2011

Journal of Biotechnology

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Section: Discussionsupporting

confidence: 92%

“…These two methods suggest a single best gene for normalisation. Recently, Feng et al (2010) have described that the worst genes in the rankings of geNorm and NormFinder were the same. Something corroborated by data shown in Figs.…”

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for gene expression studies in rats

Martínez‐Beamonte

Navarro

Larraga

et al. 2011

Journal of Biotechnology

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“…GAPDH was also reported to be relatively unstable within (Svobodová et al, 2008) and across (Nygard et al, 2007) various pig tissues. GAPDH showed least expression stability during skeletal muscle development (Feng et al, 2010) too.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the stability of ICGs across adipogenic process has not been evaluated in MSVCs. In pig, several reference gene studies have been performed in various breeds (Park et al, 2015), within (Svobodová et al, 2008) and across tissues (Nygard et al, 2007), time-course during early embryonic development (Kuijk et al, 2007), pre and postnatal skeletal muscle development (Feng et al, 2010), yielding various ICGs in different context. These results suggest that constitutively expressed ICGs are not consistently stable across all cell types, tissues and developmental stages.…”

Section: Discussionmentioning

confidence: 99%

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

Huang

Chen

et al. 2016

Animal

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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.

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“…PCRs were performed using gene-specific primer pairs (Table 1). Gene expression levels were quantified relatively to the expression of the three reference genes [36], peptidylprolyl isomerase A (PPIA), hypoxanthine phosphoribosyltransferase (HPRT) and eukaryotic elongation factor 1 gamma-like protein (eEF-1γ), using Gene Expression Macro software (ABI, USA) by employing an optimized comparative Ct (ΔΔCt) value method. The expression level was calculated as 2^(−ΔΔCt) to compare the relative expression.…”

Section: Cdna Synthesis and Qrt-pcr Analysismentioning

confidence: 99%