Czech J. Anim. Sci., 2017, 62(10):426-434 | DOI: 10.17221/75/2016-CJAS

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

Mingna Li, Xiaoyun Wu, Xian Guo, Pengjia Bao, Xuezhi Ding, Min Chu, Chunnian Liang, Ping Yan*
Key Laboratory for Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, P.R. China

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.

Keywords: transcription stability; RT-qPCR; TBP gene; ACTB gene

Published: October 31, 2017  Show citation

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Li M, Wu X, Guo X, Bao P, Ding X, Chu M, et al.. Identification of optimal reference genes for examination of gene expression in different tissues of fetal yaks. Czech J. Anim. Sci. 2017;62(10):426-434. doi: 10.17221/75/2016-CJAS.
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    References

    1. Andersen C.L., Jensen J.L., Orntoft 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. Cancer Research, 64, 5245-5250. Go to original source... Go to PubMed...
    2. Chen I., Chou L., Chou S., Wang J., Stott J., Blanchard M., Jen I.F., Yang W. (2015): Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus). Scientific Reports, 5: 15425. Go to original source... Go to PubMed...
    3. Das R.K., Banerjee S., Shapiro B.H. (2013): Extensive sexand/or hormone-dependent expression of rat housekeeping genes. Endocrine Research, 38, 105-111. Go to original source... Go to PubMed...
    4. 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. Go to original source... Go to PubMed...
    5. Huggett J., Dheda K., Bustin S., Zumla A. (2005): Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 6, 279-284. Go to original source... Go to PubMed...
    6. Jiang M., Lee J.N., Bionaz M., Deng X., Wang Y. (2016): Evaluation of suitable internal control genes for RT-qPCR in yak mammary tissue during the lactation cycle. PLoS ONE, 11, e0147705. Go to original source... Go to PubMed...
    7. Kubista M., Andrade J.M., Bengtsson M., Forootan A., Jonak J., Lind K., Sindelka R., Sjoback R., Sjofreen B., Strombom L., Stahlberg A., Zoric N. (2006): The realtime polymerase chain reaction. Molecular Aspects of Medicine, 27, 95-125. Go to original source... Go to PubMed...
    8. Lisowski P., Pierzchala M., Goscik J., Pareek C.S., Zwierzchowski L. (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. Go to original source... Go to PubMed...
    9. Liu B., Cui Y., Yang B., Fan J., Zhao Z., Yu S. (2010): Morphometric analysis of yak placentomes during gestation. Anatomical Record (Hoboken), 293, 1873-1879. Go to original source... Go to PubMed...
    10. Liu J., Wang Q., Sun M., Zhu L., Yang M., Zhao Y. (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. Go to original source... Go to PubMed...
    11. Martianov I., Viville S., Davidson I. (2002): RNA polymerase II transcription in murine cells lacking the TATA binding protein. Science, 298, 1036-1039. Go to original source... Go to PubMed...
    12. Mihi B., Rinaldi M., Geldhof P. (2011): Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum. Veterinary Parasitology, 181, 354-359. Go to original source... Go to PubMed...
    13. Muller F., Lakatos L., Dantonel J., Strahle U., Tora L. (2001): TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Current Biology, 11, 282-287. Go to original source... Go to PubMed...
    14. Nygard A.B., Jorgensen C.B., Cirera S., Fredholm M. (2007): Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology, 8: 67. Go to original source... Go to PubMed...
    15. Pan Y., He H., Cui Y., Baloch A.R., Li Q., Fan J., He J., Yu S. (2015): Recombinant human bone morphogenetic protein 6 enhances oocyte reprogramming potential and subsequent development of the cloned yak embryos. Cellular Reprogramming, 17, 483-493. Go to original source... Go to PubMed...
    16. Pfaffl M.W., Tichopad A., Prgomet C., Neuvians T.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. Go to original source... Go to PubMed...
    17. Ponomarenko M.P., Arkova O., Rasskazov D., Ponomarenko P., Savinkova L., Kolchanov N. (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: 130. Go to original source... Go to PubMed...
    18. Purohit G.K., Mahanty A., Mohanty B.P., Mohanty S. (2015): 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. Go to original source... Go to PubMed...
    19. Radonic A., Thulke S., Mackay I.M., Landt O., Siegert W., Nitsche A. (2004): Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313, 856-862. Go to original source... Go to PubMed...
    20. Rekawiecki R., Rutkowska J., Kotwica J. (2012): Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum. Reproductive Biology, 12, 362-367. Go to original source... Go to PubMed...
    21. Sakai H., Sato K., Kai Y., Shoji T., Hasegawa S., Nishizaki M., Sagara A., Yamashita A., Narita M. (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. Go to original source... Go to PubMed...
    22. Schmittgen T.D., Livak K.J. (2008): Analyzing real-time PCR data by the comparative C T method. Nature Protocols, 3, 1101-1108. Go to original source... Go to PubMed...
    23. 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. Go to original source... Go to PubMed...
    24. Svingen T., Letting H., Hadrup N., Hass U., Vinggaard A.M. (2015): Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ Computer Science, 3: e855. Go to original source... Go to PubMed...
    25. 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.
    26. 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. Go to original source...
    27. Wu X., Ding X., Chu M., Guo X., Bao P., Liang C., Yan P. (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. Go to original source...
    28. Yang C., Pan H., Noland J.E., Zhang D., Zhang Z., Liu Y., Zhou X. (2015): Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific Reports, 5: 18201. Go to original source... Go to PubMed...
    29. 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. Go to original source... Go to PubMed...
    30. Zeng J., Liu S., Zhao Y., Tan X., Aljohi H.A., Liu W., Hu S. (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. Go to original source... Go to PubMed...
    31. Zhang Y., Zhang X.D., Liu X., Li Y.S., Ding J.P., Zhang X.R., Zhang Y.H. (2013): Reference gene screening for analyzing gene expression across goat tissue. Asian-Australasian Journal of Animal Sciences, 26, 1665-1671. Go to original source... Go to PubMed...

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