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

Published online by Cambridge University Press:  19 January 2016

X. Li ,
K. Huang ,
F. Chen ,
W. Li ,
S. Sun ,
X.-E. Shi  and
G. Yang
X. Li
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
K. Huang
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
F. Chen
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
W. Li
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
S. Sun
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
X.-E. Shi
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
G. Yang*
Affiliation:
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R.China
*
†E-mail: gsyang999@hotmail.com
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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.

Keywords

reference genequantitative real-time PCRpigintramuscular stromal-vascular cellsintramuscular fat
Type
Research Article
Information
animal , Volume 10 , Issue 6 , June 2016 , pp. 947 - 952
Copyright
© The Animal Consortium 2016 

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Footnotes

a

Both authors contributed equally to this work.

References

Andersen, CL, Jensen, JL and Ørntoft, TF 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64, 5245–5250.CrossRefGoogle Scholar
Ayuso, M, Óvilo, C, Rodríguez-Bertos, A, Rey, AI, Daza, A, Fenández, A, González-Bulnes, A, López-Bote, CJ and Isabel, B 2015. Dietary vitamin A restriction affects adipocyte differentiation and fatty acid composition of intramuscular fat in Iberian pigs. Meat Science 108, 9–16.CrossRefGoogle ScholarPubMed
Bionaz, M and Loor, JJ 2007. Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle. Physiological Genomics 29, 312–319.CrossRefGoogle ScholarPubMed
Erkens, T, Van Poucke, M, Vandesompele, J, Goossens, K, Van Zeveren, A and Peelman, LJ 2006. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A. BMC Biotechnology 6, 41.CrossRefGoogle ScholarPubMed
Feng, X, Xiong, Y, Qian, H, Lei, M, Xu, D and Ren, Z 2010. Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR. Journal of Biotechnology 150, 288–293.CrossRefGoogle ScholarPubMed
Garibyan, L and Avashia, N 2013. Research techniques made simple: polymerase chain reaction (PCR). The Journal of Investigative Dermatology 133, e6.Google Scholar
Guénin, S, Mauriat, M, Pelloux, J, Van Wuytswinkel, O, Bellini, C and Gutierrez, L 2009. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. Journal of Experimental Botany 60, 487–493.CrossRefGoogle ScholarPubMed
Guo, Y, Mo, D, Zhang, Y, Zhang, Y, Cong, P, Xiao, S, He, Z, Liu, X and Chen, Y 2012. MicroRNAome comparison between intramuscular and subcutaneous vascular stem cell adipogenesis. PLoS ONE 7, e45410.CrossRefGoogle ScholarPubMed
Hausman, G and Poulos, S 2004. Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles. Journal of Animal Science 82, 429–437.CrossRefGoogle ScholarPubMed
Hocquette, JF, Gondret, F, Baéza, E, Médale, F, Jurie, C and Pethick, DW 2010. Intramuscular fat content in meat-producing animals: development, genetic and nutritional control, and identification of putative markers. Animal 4, 303–319.Google Scholar
Ji, N, Li, L, Lin, L and Lin, S 2015. Screening for suitable reference genes for quantitative real-time PCR in Heterosigma akashiwo (Raphidophyceae). PLoS ONE 10, e0132183.Google Scholar
Jiang, S, Wei, H, Song, T, Yang, Y, Peng, J and Jiang, S 2013. Transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation. PLoS ONE 8, e77094.Google Scholar
Kuijk, E, du Puy, L, van Tol, H, Haagsman, H, Colenbrander, B and Roelen, B 2007. Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Developmental Biology 7, 58.Google Scholar
Li, R and Shen, Y 2013. An old method facing a new challenge: re-visiting housekeeping proteins as internal reference control for neuroscience research. Life Sciences 92, 747–751.CrossRefGoogle ScholarPubMed
Liang, Y, Yang, X, Gu, Y, Tao, X, Zhong, Z, Gong, J, Chen, X and Lv, X 2015. Developmental changes in the expression of the GLUT2 and GLUT4 genes in the longissimus dorsi muscle of Yorkshire and Tibetan pigs. Genetic and Molecular Research 14, 1287–1292.Google Scholar
Monaco, E, Bionaz, M, de Lima, AS, Hurley, WL, Loor, JJ and Wheeler, MB 2010. Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells. Stem Cell Research & Therapy 1, 7.CrossRefGoogle ScholarPubMed
Nygard, A-B, Jørgensen, CB, Cirera, S and Fredholm, M 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology 8, 67.Google Scholar
Park, S-J, Kwon, SG, Hwang, JH, Park, DH, Kim, TW and Kim, CW 2015. Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs. Gene 558, 152–158.Google Scholar
Pfaffl, M, Tichopad, A, Prgomet, C and Neuvians, T 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters 26, 509–515.Google Scholar
Pu, Y, Guo, B, Liu, D, Xiong, H, Wang, Y and Du, H 2015. Iron supplementation attenuates the inflammatory status of anemic piglets by regulating hepcidin. Biological Trace Element Research 167, 1–8.Google Scholar
Svobodová, K, Bílek, K and Knoll, A 2008. Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig. Journal of Applied Genetics 49, 263–265.CrossRefGoogle ScholarPubMed
Vandesompele, J, De Preter, K, Pattyn, F, Poppe, B, Van Roy, N, De Paepe, A and Speleman, F 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biology 3, research0034.Google Scholar
Wang, G-Q, Zhu, L, Ma, M-L, Chen, X-C, Gao, Y, Yu, T-Y, Yang, G-S and Pang, W-J 2015. Mulberry 1-deoxynojirimycin inhibits adipogenesis by repression of the ERK/PPARγ signaling pathway in porcine intramuscular adipocytes. Journal of Agricultural and Food Chemistry 63, 6212–6220.CrossRefGoogle ScholarPubMed
Wood, JD, Enser, M, Fisher, AV, Nute, GR, Sheard, PR, Richardson, RI, Hughes, SI and Whittington, FM 2008. Fat deposition, fatty acid composition and meat quality: a review. Meat Science 78, 343–358.Google Scholar
Zhang, J, Tang, Z, Wang, N, Long, L and Li, K 2012. Evaluating a set of reference genes for expression normalization in multiple tissues and skeletal muscle at different development stages in pigs using quantitative real-time polymerase chain reaction. DNA and Cell Biology 31, 106–113.CrossRefGoogle ScholarPubMed
Zhang, Y, Li, D and Sun, B 2015. Do housekeeping genes exist? PLoS ONE 10, e0123691.Google Scholar
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