Identification of optimal reference genes for examination of gene expression in different tissues of fetal yaks

DOI:10.17221/75/2016-CJASCitation:Li M., Wu X., Guo X., Bao P., Ding X., Chu M., Liang C., Yan P. (2017): Identification of optimal reference genes for examination of gene expression in different tissues of fetal yaks. Czech J. Anim. Sci., 62: 426-434.
download PDF

Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used to study the relative abundance of mRNA transcripts because of its sensitivity and reliable quantification. However, the reliability of the interpretation of expression data is influenced by several complex factors, including RNA quality, transcription activity, and PCR efficiency, among others. To avoid experimental errors arising from potential variation, the selection of appropriate reference genes to normalize gene expression is essential. In this study, 10 commonly used reference genes – ACTB, B2M, HPRT1, GAPDH, 18SrRNA, 28SrRNA, PPIA, UBE2D2, SDHA, and TBP – were selected as candidate reference genes for six fetal tissues (heart, liver, spleen, lung, kidney, and forehead skin) of yak (Bos grunniens). The transcription stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper. The results showed that the combination of TBP and ACTB provided high-quality data for further study. In contrast, the commonly used reference genes 28SrRNA, SDHA, GAPDH, and B2M should not be used for endogenous controls because of their unstable expression in this study. The reference genes that could be used in future gene expression studies in yaks were indentified.

Andersen Claus Lindbjerg, Jensen Jens Ledet, Ørntoft Torben Falck (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 doi:10.1158/0008-5472.CAN-04-0496
Chen I-Hua, Chou Lien-Siang, Chou Shih-Jen, Wang Jiann-Hsiung, Stott Jeffrey, Blanchard Myra, Jen I-Fan, Yang Wei-Cheng (2015): Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus). Scientific Reports, 5, - doi:10.1038/srep15425
Das Rajat K., Banerjee Sarmistha, Shapiro Bernard H. (2013): Extensive Sex- and/or Hormone-Dependent Expression of Rat Housekeeping Genes. Endocrine Research, 38, 105-111 doi:10.3109/07435800.2012.723294
Hu R., Wang Z., Peng Q., Zou H., Wang H., Yu X., Jing X., Wang Y., Cao B., Bao S., Zhang W., Zhao S., Ji H., Kong X., Niu Q. (2016): Effects of GHRP-2 and cysteamine administration on growth performance, somatotropic axis hormone and muscle protein deposition in yaks (Bos grunniens) with growth retardation. PLoS ONE, 11, e0149461.
Huggett J, Dheda K, Bustin S, Zumla A (2005): Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 6, 279-284 doi:10.1038/sj.gene.6364190
Jiang MingFeng, Lee Jung Nam, Bionaz Massimo, Deng Xiao Yu, Wang Yong, Seagroves Tiffany (2016): Evaluation of Suitable Internal Control Genes for RT-qPCR in Yak Mammary Tissue during the Lactation Cycle. PLOS ONE, 11, e0147705- doi:10.1371/journal.pone.0147705
Kubista Mikael, Andrade José Manuel, Bengtsson Martin, Forootan Amin, Jonák Jiri, Lind Kristina, Sindelka Radek, Sjöback Robert, Sjögreen Björn, Strömbom Linda, Ståhlberg Anders, Zoric Neven (2006): The real-time polymerase chain reaction. Molecular Aspects of Medicine, 27, 95-125 doi:10.1016/j.mam.2005.12.007
Lisowski Paweł, Pierzchała Mariusz, Gościk Joanna, Pareek Chandra S., Zwierzchowski Lech (2008): Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid. Journal of Applied Genetics, 49, 367-372 doi:10.1007/BF03195635
Liu Ben, Cui Yan, Yang Bo, Fan Jiangfeng, Zhao Zhiwen, Yu Sijiu (2010): Morphometric Analysis of Yak Placentomes During Gestation. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, 293, 1873-1879 doi:10.1002/ar.21231
Liu Jing, Wang Qun, Sun Minying, Zhu Linlin, Yang Michael, Zhao Yu, Dettman Robert W. (2014): Selection of Reference Genes for Quantitative Real-Time PCR Normalization in Panax ginseng at Different Stages of Growth and in Different Organs. PLoS ONE, 9, e112177- doi:10.1371/journal.pone.0112177
Martianov I., Viville S., Davidson I. (2002): RNA Polymerase II Transcription in Murine Cells Lacking the TATA Binding Protein. Science, 298, 1036-1039 doi:10.1126/science.1076327
Mihi Belgacem, Rinaldi Manuela, Geldhof Peter (2011): Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum. Veterinary Parasitology, 181, 354-359 doi:10.1016/j.vetpar.2011.04.009
Müller Ferenc, Lakatos Lòrànt, Dantonel Jean-Christophe, Strähle Uwe, Tora Làszlò (2001): TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Current Biology, 11, 282-287 doi:10.1016/S0960-9822(01)00076-8
Nygard Ann-Britt, Jørgensen Claus B, Cirera Susanna, Fredholm Merete (): Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology, 8, 67- doi:10.1186/1471-2199-8-67
Pan Yangyang, He Honghong, Cui Yan, Baloch Abdul Rasheed, Li Qin, Fan Jiangfeng, He Junfeng, Yu Sijiu (2015): Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos. Cellular Reprogramming, 17, 484-493 doi:10.1089/cell.2015.0049
Pfaffl Michael W., Tichopad Ales, Prgomet Christian, Neuvians Tanja P. (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 doi:10.1023/B:BILE.0000019559.84305.47
Ponomarenko Mikhail P., Arkova Olga, Rasskazov Dmitry, Ponomarenko Petr, Savinkova Ludmila, Kolchanov Nikolay (2016): Candidate SNP Markers of Gender-Biased Autoimmune Complications of Monogenic Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters. Frontiers in Immunology, 7, - doi:10.3389/fimmu.2016.00130
Purohit Gopal Krishna, Mahanty Arabinda, Mohanty Bimal Prasanna, Mohanty Sasmita (2016): Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress. Fish Physiology and Biochemistry, 42, 125-135 doi:10.1007/s10695-015-0123-0
Radonić Aleksandar, Thulke Stefanie, Mackay Ian M, Landt Olfert, Siegert Wolfgang, Nitsche Andreas (2004): Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313, 856-862 doi:10.1016/j.bbrc.2003.11.177
Rekawiecki Robert, Rutkowska Joanna, Kotwica Jan (2012): Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum. Reproductive Biology, 12, 362-367 doi:10.1016/j.repbio.2012.10.010
Sakai Hiroyasu, Sato Ken, Kai Yuki, Shoji Tetsuro, Hasegawa Satoshi, Nishizaki Maiko, Sagara Atsunobu, Yamashita Akira, Narita Minoru (2014): Distribution of aquaporin genes and selection of individual reference genes for quantitative real-time RT-PCR analysis in multiple tissues of the mouse. Canadian Journal of Physiology and Pharmacology, 92, 789-796 doi:10.1139/cjpp-2014-0157
Schmittgen Thomas D, Livak Kenneth J (2008): Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101-1108 doi:10.1038/nprot.2008.73
Spinsanti G., Panti C., Lazzeri E., Marsili L., Casini S., Frati F., Fossi C.M. (2006): Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Molecular Biology, 7: 32.doi:10.1186/1471-2199-7-32
Svingen Terje, Letting Heidi, Hadrup Niels, Hass Ulla, Vinggaard Anne Marie (2015): Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ, 3, e855- doi:10.7717/peerj.855
Touchberry C.D., Wacker M J., Richmond S.R., Whitman S.A., Godard M.P. (2006): Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle. Journal of Biomolecular Techniques, 17, 157–162.
Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. (2002): Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3: research0034.1.
WU Xiao-yun, DING Xue-zhi, CHU Min, GUO Xian, BAO Peng-jia, LIANG Chun-nian, YAN Ping (2015): Novel SNP of EPAS1 gene associated with higher hemoglobin concentration revealed the hypoxia adaptation of yak (Bos grunniens). Journal of Integrative Agriculture, 14, 741-748 doi:10.1016/S2095-3119(14)60854-6
Yang Chunxiao, Pan Huipeng, Noland Jeffrey Edward, Zhang Deyong, Zhang Zhanhong, Liu Yong, Zhou Xuguo (2016): Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific Reports, 5, - doi:10.1038/srep18201
Young N.J., Thomas C.J., Collins M.E., Brownlie J. (2006): Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference. Journal of Virological Methods, 138, 218-222 doi:10.1016/j.jviromet.2006.08.008
Zeng Jingyao, Liu Shoucheng, Zhao Yuhui, Tan Xinyu, Aljohi Hasan Awad, Liu Wanfei, Hu Songnian (2016): Identification and analysis of house-keeping and tissue-specific genes based on RNA-seq data sets across 15 mouse tissues. Gene, 576, 560-570 doi:10.1016/j.gene.2015.11.003
Zhang Yu, Zhang Xiao-Dong, Liu Xing, Li Yun-Sheng, Ding Jian-Ping, Zhang Xiao-Rong, Zhang Yun-Hai (2013): Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue. Asian-Australasian Journal of Animal Sciences, 26, 1665-1671 doi:10.5713/ajas.2013.13199
download PDF

© 2018 Czech Academy of Agricultural Sciences