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Screening of Reference Genes for qRT-PCR in Bama Minipig (Sus scrofa) Tissues |
CHEN Chu-Jie1, PEI Yang-Li1,*, LIU Lu-Lu2, YANG Ya-Lan1, ZHANG Lei-Xia1, LI Hua1, LI Kui1,2 |
1 Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding/School of Life Sciences and Engineering, Foshan University, Foshan 528225, China; 2 Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China |
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Abstract qRT-PCR is one of the important methods for investigating mRNA expression levels in the cells and tissues. However, the selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. This study examined the expression of 9 internal genes (topoisomerase (DNA)Ⅱβ (Top2b), β-actin (Actb), ribosomal protein S18 (Rps18), peptidylprolyl isomerase A (Ppia), 18S ribosomal RNA (18S), TATA-box binding protein (Tbp), hydroxymethylbilane synthase (Hmbs), ribosomal protein L4 (Rpl4) and β-2-microglobulin (B2m) for heart, liver, spleen, lung, kidney, muscle, subcutaneous fat from three 15 d Bama minipigs (Sus scrofa) using qRT-PCR technique, and 3 well-known software packages: geNorm, NormFinder and BestKeeper were used to analyze the results. Rps18, Rpl4, Ppia and Tbp were found to own the highest stability across tissues. Only 2 internal reference genes could standardize the expression of target genes in the same tissue. And in the different tissue, the optimal gene combination was different. The results of this study provide a reference information on Bama minipig to select the most suitable internal reference gene, and it could provide a theoretical basis for the research of gene function about meat quality, litter size, development, reproduction, disease and so on of Bama minipig.
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Received: 24 December 2019
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Corresponding Authors:
*, peiyangli@163.com
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[1] 庞琳琳, 张会永, 杨关林. 2014. 巴马小型猪在医学研究中的应用进展[J]. 中国实验动物学报, 22(01): 94-98. (Pang L L, Zhang H Y, Yang G L.2014. Application of Chinese Bama minipigs in medical research: A literature review[J]. Acta Laboratorium Animalis Scientia Sinica, 22(01): 94-98) [2] 赵晶, 赵俊杰, 康丽丽,等. 2014. 人不同孕期胎盘组织中实时荧光定量PCR内参基因的选择[J]. 西安交通大学学报(医学版), 35(02): 218-221. (Zhao J, Zhao J J, Kang L L, et al.2014. Selection of suitable reference genes for quantitative real-time PCR in human villi and placenta of different pregnancy[J]. Journal of Xi'an Jiaotong University (Medical Sciences), 35(02): 218-221.) [3] 翟斐, 杨秉芬, 王若, 等. 2018. 活动性肺结核患者PBMCs实时定量PCR内参基因的选择[J]. 临床肺科杂志, 23(11): 1941-1945+1958. (Di F, Yang B F, Wang R, et al. 2018. Selection of suitable reference genes for quantitative real-time PCR in PBMCs from active pulmonary tuber-culosis patients[J]. Journal of Clinical Pulmonary Medicine, 23(11): 1941-1945+1958.) [4] 台玉磊, 韩立强, 杨国庆, 等. 2010. 仔猪组织基因表达中实时定量PCR内参基因的选择[J]. 农业生物技术学报, 18(04): 732-736. (Tai Y L, Han L Q, Yang G Q, et al.2010. Selection of the reference genes of real-time quantitative PCR in the gene expression of piglet tissues[J]. Journal of Agricultural Biotechnology, 18(04): 732-736.) [5] 彭馥芝, 冉茂良, 翁波, 等. 2017. 猪睾丸组织定量PCR分析中内参基因的选择[J]. 中国农业科学, 50(15): 3033-3041. (Peng F Z, Rang M L, Weng B, et al.2017. Validation of reference genes for quantitative RT-PCR analysis in porcine testis tissues[J]. Scientia Agricultura Sinica, 50(15): 3033-3041.) [6] 冯小婷. 2011. 梅山—大白猪肌肉组织差异表达基因的筛选、鉴定及功能研究[D]. 博士学位论文, 华中农业大学, 导师: 熊远著. pp. 1-122. (Feng X T.2011. Thesis for Ph.D. HuaZhong Agricultural University, Supervisor:Xiong Y Z, pp. 1-122.) [7] 齐波, 朱荣生, 王怀中, 等. 2017. 测定猪不同组织基因表达时RT-PCR内参基因的选择[J]. 家畜生态学报, 2017(06): 14-18. (Qi B, Zhu R S, Wang H Z, et al.2017. Selection of reference genes of real-time quantitative PCR in the gene expression of different tissues in pig[J]. Acta Ecologae Animalis Domastici, 2017(06): 14-18.) [8] Andersen C L, Jensen J L, Ørntoft T F.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[J]. Cancer Research, 64(15): 5245-5250. [9] Baumann A, Rüdiger Lehmann, Beckert A, et al.2015. Selection and evaluation of tissue specific reference genes in lucilia sericata during an immune challenge[J]. PLOS ONE, 10(8): e0135093. [10] Bonnet M, Bernard L, Bes S, et al.2013. Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants[J]. Animal, 7(08): 1344-1353. [11] Bruun C S, Jensen L K, Páll Skuli Leifsson, et al.2013. Functional Characterization of a porcine emphysema model[J]. Lung, 191(6): 669-675. [12] Bronkhorst A, Aucamp J, Wentzel J, et al.2016. Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling[J]. Clinical Biochemistry, 49(7-8): 606-608. [13] Guo Y, Pennell M, Pearl D, et al.2013. The choice of reference gene affects statistical efficiency in quantitative PCR data analysis[J]. Biotechniques, 55(4): 207-209. [14] Huggett J, Dheda K, Bustin S, et al.2005. Real-time RT-PCR normalisation; strategies and considerations[J]. Genes and Immunity, 6(4): 279-284. [15] Jain M, Nijhawan A, Tyagi A K, et al.2006. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 345(2): 646-651. [16] Jian B, Liu B, Yurong B, et al.2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J]. BMC Molecular Biology, 9(1): 1-14. [17] Kosir R, Acimovic J, Golicnik M, et al.2010. Determination of reference genes for circadian studies in different tissues and mouse strains[J]. BMC Molecular Biology, 11(1): 60. [18] Liu Y, Zeng B H, Shang H T, et al.2008. Bama miniature pigs (Sus scrofa domestica) as a model for drug evaluation for humans: Comparison of in vitro metabolism and in vivo pharmacokinetics of lovastatin[J]. Comparative Medicine, 58(6): 580-587. [19] Mackay I M .2004. Real-time PCR in the microbiology laboratory[J]. Clinical Microbiology and Infectious, 10(3): 190-212. [20] Martino A, Cabiati M, Campan M, et al.2011. Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-uropean Society of Cl[J]. Journal of Biotechnology, 153(3-4): 92-99. [21] Nygard A B, Claus B Jørgensen, Cirera S, et al.2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR[J]. BMC Molecular Biology, 8(1):67. [22] Park S J, Huh J W, Kim Y H, et al.2013. Selection of internal reference genes for normalization of quantitative;reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs[J]. Molecular Biotechnology, 54(1): 47-57. [23] Park S J, Kwon S G, Hwang J H, et al.2015. Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs[J]. Gene, 558(1): 152-158. [24] Pfaffl M W, Tichopad A, Prgomet C, et al.2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 26(6): 509-515. [25] Pfister C, Tatabiga M S, Roser F.2011. Selection of suitable reference genes for quantitative real-time polymerase chain reaction in human meningiomas and arachnoidea[J]. BMC research notes, 4: 275. [26] Qiao S, Deng Y, Li S, et al.2019. Partially reprogrammed induced pluripotent stem cells using microRNA cluster miR-302s in Guangxi Bama minipig fibroblasts[J]. Cellular Reprogramming, 21(5): 229-237. [27] Shang H, Guo K, Liu Y, et al.2013. Constitutive expression of CYP3A mRNA in Bama miniature pig tissues[J]. Gene, 524(2): 261-267. [28] Sumby K M, Grbin P R, Jiranek V.2012. Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni[J]. Applied Microbiology and Biotechnology, 96(4): 1039-1047. [29] Svobodová K, Bílek K, Knoll A.2008. Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig[J]. Journal of Applied Genetics, 49(3): 263-265. [30] Tang Z, Li Y, Wan P, et al.2007. LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs[J]. Genome Biology, 8(6): R115. [31] Valasek M A, Repa J J.2005. The power of real-time PCR[J]. AJP: Advances in Physiology Education, 29(3): 151. [32] Vandesompele J, De Preter K, Pattyn F, et al.2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 3(7): research0034.1. [33] Wang G, Chen Y, Zhang X, et al.2018. Selection of reference genes for tissue/organ samples on day 3 fifth-instar larvae in silkworm, Bombyx mori[J]. Archives of Insect Biochemistry and Physiology, 98(2): e21458. [34] Wang N N, Chen G N, Qu B, et al.2019a. Effect of Hypotensive Brain Death on the Donor Liver and Its Mechanism in an Improved Bama Miniature Pig (Sus scrofa domestica) Model[J]. Transplantation Proceeding, 51(3): 951-959. [35] Wang R, Zhang J Y, Lu K H, et al.2019b. Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing[J]. In vitro Cellular & Developmental Biology. Animal. [36] Wang Y, Zhao Y, Li J, et al.2015. Evaluation of housekeeping genes for normalizing real-time quantitative PCR assays in pig skeletal muscle at multiple developmental stages[J]. Gene, 565(2): 235-241. [37] Wierschke S, Gigout S, Horn P, et al.2010. Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues[J]. Biochemical & Biophysical Research Communications, 403(3-4): 385-390. [38] Zhang Y, Zhang X D, Liu X, et al.2013. Reference gene screening for analyzing gene expression across goat tissue[J]. Asian-australasian Journal of Animal Sciences, 26(12): 1665-1671. [39] Zhou X, Liu J, Zhuang Y.2014. Selection of appropriate reference genes in eggplant for quantitative gene expression studies under different experimental conditions[J]. Scientia Horticulturae, 176(2): 200-207. |
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