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PU Fei, YANG Bingye, KE Caihuan. Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata[J]. Journal of Oceanology and Limnology, 2015, 33(4): 838-845

Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

PU Fei1,2, YANG Bingye2,3, KE Caihuan1,2
1 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, China;
2 College of Ocean and Earth Science, Xiamen University, Xiamen 361102, China;
3 Xiamen Medical College, Xiamen 361008, China
Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene () across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.
Key words:    Crassostrea angulata|gene expression|quantitative real-time PCR|internal control gene|G protein β-subunit gene   
Received: 2014-03-22   Revised: 2014-11-11
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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. Cancer Research, 64 (15): 5 245- 5 250.
Araya M T, Siah A, Mateo D, Markham F, McKenna P, Johnson G, Berthe F C J. 2008. Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus. Journal of Invertebrate Pathology, 99 (3): 326-331.
Aursnes I A, Rishovd A L, Karlsen H E, Gjøen T. 2011. Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua L.) during temperature stress. BMC Research Notes, 4 : 104.
Bustin S A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology, 29 (1): 23-29.
Castro L F C, Melo C, Guillot R, Mendes I, Queirós S, Lima D, Reis-Henriques M A, Santos M M. 2007. The estrogen receptor of the gastropod (Nucella lapillus): modulation following exposure to an estrogenic effluent? Aquatic Toxicology, 84 (4): 465-468.
Chen L, Xie L P, Xiong X H, Dai Y P, Fan W M, Zhang R Q. 2005. Cloning and characterization of a novel G protein β-subunit of pearl oyster (Pinctada fucata), and its interaction sites with calmodulin. Comparative Biochemistry and Physiology Part B : Biochemistry & Molecular Biology, 142 (2): 142-152.
Cho Y S, Lee S Y, Kim K H, Nam Y K. 2008. Differential modulations of two glyceraldehyde 3-phosphate dehydrogenase mRNAs in response to bacterial and viral challenges in a marine teleost Oplegnathus fasciatus (Perciformes). Fish & Shellfish Immunology, 25 (5): 472- 476.
Cubero-Leon E, Ciocan C M, Minier C, Rotchell J M. 2011. Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure. Environmental Science and Pollution Research, 19 (7): 2 728-2 733.
Dheilly N M, Lelong C, Huvet A, Favrel P. 2011. Development of a Pacific oyster (Crassostrea gigas) 31, 918-feature microarray: identification of reference genes and tissueenriched expression patterns. BMC Genomics, 12 (1): 468.
Dondero F, Dagnino A, Jonsson H, Caprì F, Gastaldi L, Viarengo A. 2006. Assessing the occurrence of a stress syndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach. Aquatic Toxicology, 78 (1): S13-S24.
Feng L Y, Yu Q, Li X, Ning X H, Wang J, Zou J J, Zhang L L, Wang S, Hu J J, Hu X L, Bao Z M. 2013. Identification of reference genes for qRT-PCR analysis in Yesso scallop Patinopecten yessoensis. PloS One, 8 (9): e75609.
Janská A, Hodek J, Svoboda P, Zámečník J, Prášil I T, Vlasáková E, Milella L, Ovesná J. 2013. The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress. Molecular Genetics and Genomics, 288 (11): 639-649.
Lamsam-Casalotti S, Onoda M, Papadopoulos V, Dym M. 1993. Developmental expression of GTP-binding proteins in rat testes. Journal of Reproduction and Fertility, 99 (2): 487-495.
Leelatanawit R, Klanchui A, Uawisetwathana U, Karoonuthaisiri N. 2012. Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp. PloS One, 7 (12): e52677.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 25 (4): 402-408.
Llera-Herrera R, García-Gasca A, Huvet A, Ibarra A M. 2012. Identification of a tubulin -α gene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus. Marine Genomics, 6 : 33- 44.
Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M. 2010. Validation of reference genes for quantitative realtime PCR during leaf and flower development in Petunia hybrida. BMC Plant Biology, 10 (1): 4.
Morga B, Arzul I, Faury N, Renault T. 2010. Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR. Fish & Shellfish Immunology, 29 (6): 937-945.
Niesters H G M. 2001. Quantitation of viral load using realtime amplification techniques. Methods, 25 (4): 419-429.
Nygard A B, Jørgensen 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 (1): 67.
Olsvik P A, Lie K K, Jordal A E, Nilsen T O, Hordvik I. 2005. Evaluation of potential reference genes in real-time RTPCR studies of Atlantic salmon. BMC Molecular Biology, 6 : 21.
PfafflM W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res., 29 (9): e45.
Setiawan A N, Lokman P M. 2010. The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale. BMC Molecular Biology, 11 (1): 75.
Siah A, Dohoo C, McKenna P, Delaporte M, Berthe F. 2008. Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria. Fish & Shellfish Immunology, 25 (3): 202-207.
Ståhlberg A, Åman P, Ridell B, Mostad P, Kubista M. 2003. Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of κ and λ immunoglobulin light chain expression. Clinical Chemistry, 49 (1): 51-59.
Sternberg R M, Hotchkiss A K, Leblanc G A. 2008. The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Ilyanassa obsoleta. General and Comparative Endocrinology, 156 (1): 15-26.
Tang R, Dodd A, Lai D, McNabb W C, Love D R. 2007. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochimica et Biophysica Sinica, 39 (5): 384-390.
Thellin O, ElMoualij B, Heinen E, Zorzi W. 2009. A decade of improvements in quantification of gene expression and internal standard selection. Biotechnology Advances, 27 (4): 323-333.
Tsugama D, Liu S, Takano T. 2013. Arabidopsis heterotrimeric G protein βsubunit, AGB1, regulates brassinosteroid signalling independently of BZR1. Journal of Experimental Botany, 64 (11): 3 213-3 223.
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 (7), research0034.
Vorachek W R, Hugejiletu, Bobe G, Hall J A. 2013. Reference gene selection for quantitative PCR studies in sheep neutrophils. International Journal of Molecular Sciences, 14 (6): 11 484-11 495.
Vu H L, Troubetzkoy S, Nguyen H H, Russell M W, Mestecky J. 2000. A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis. Nucleic Acids Res., 28 (7): e18.
Wang T, Liang Z A, Sandford A J, Xiong X Y, Yang Y Y, Ji Y L, He J Q. 2012. Selection of suitable housekeeping genes for real-time quantitative PCR in CD 4+ lymphocytes from asthmatics with or without depression. PloS One, 7 (10): e48367.
Watson A J, Katz A, Simon M I. 1994. A fifth member of the mammalian G-protein β-subunit family. expression in brain and activation of the β2 isotype of phospholipase C. The Journal of Biological Chemistry, 269 : 22 150-22 156.
Zhai Z C, Yao Y C, Wang Y J. 2013. Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation. PloS One, 8 (5): e64786.
Zhang G, Zhao M M, Song C, Luo A X, Bai J F, Guo S X. 2012. Characterization of reference genes for quantitative realtime PCR analysis in various tissues of Anoectochilus roxburghii. Molecular Biology Reports, 39 (5): 5 905-5 912.
Zhang K, Niu S F, Di D P, Shi L D, Liu D S, Cao X L, Miao H Q, Wang X B, Han C G, Yu J L, Li D W, Zhang Y L. 2013. Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR. Journal of Biotechnology, 168 (1): 7-14.
Zhong Q W, Zhang Q Q, Wang Z G, Qi J, Chen Y J, Li S, Sun Y Y, Li C M, Lan X. 2008. Expression profiling and validation of potential reference genes during Paralichthys olivaceus embryogenesis. Marine Biotechnology, 10 (3): 310-318.
Zhu J F, Zhang L F, Li W F, Han S Y, Yang W H, Qi L W. 2013. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PloS One, 8 (1): e53196.
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