Abstract
A stable internal control gene or house keeping gene (HKG) is often used to normalise mRNA levels in different samples for expression analysis. In the present study, the authors identified and evaluated three HKGs, beta actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1 alpha (EF1α) for gene expression study in Catla (Catla catla). Gene expression levels were quantified by quantitative real-time reverse transcription polymerase chain reaction in different tissues (liver, kidney, intestine, gill, muscle and brain) and developmental stages (0, 3, 6, 12, 24 h, and 5, 7, 9 days post-fertilization). Expression stability was evaluated by comparing the coefficients of variation of the cycle threshold values and stability index. All the tested HKGs exhibited wide expression range. The results showed that β-actin is the most stable gene followed by the EF1α and GAPDH in different tissues whereas GAPDH was most stable gene followed by EF1α and β-actin during embryonic development.
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Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalization; strategies and considerations. Genes Immunol 6:279–284
Yang CG, Wang XL, Tian J, Liu W, Wu F, Jiang M, Wen H (2013) Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus). Gene 527(1):183–192
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N (eds) Real-Time PCR: current technology and applications. Caister Academic Press, London, pp 47–64
An L, Hu J, Yang M, Zheng B, Wei A, Shang J, Zhao X (2011) CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China. Mar Pollut Bull 62(4):718–725
Szabo A, Perou CM, Karaca M, Perreard L, Quackenbush JF, Bernard PS (2004) Statistical modeling for selecting housekeeper genes. Genome Biol 5:R59
Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344(1):141–143
Tang YK, Yu YH, Xu P, Li JL, Li HX, Ren HT (2012) Identification of housekeeping genes suitable for gene expression analysis in Jian carp (Cyprinus carpio var. jian). Fish Shellfish Immunol 33:775–779
Vandesompele J, Preter DK, Pattyn F, Poppe B, Roy VN, Paepe DA, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:34
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper-excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515
Andersen CL, Jensen JL, Orntoft 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 Res 64:5245–5250
Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14
Jena JK (2014) Cultured aquatic species information programme: Catla catla. FAO Fisheries and Aquaculture Department [online], Rome. http://www.fao.org/fishery/culturedspecies/Catla_catla/en. Accessed 28 Aug 2014
Uma A, Rebecca G, Saravanabava K (2012) In vivo effect of cpg oligodeoxynucleotides (CPG ODNS) on the expression of toll-like receptor9 (TLR9) in Catla catla. CIBtech J Biotechnol 1:17–21
Swain B, Samanta M, Basu M, Panda P, Sahoo BR, Maiti NK, Mishra BK, Eknath AE (2012) Molecular characterization, inductive expression and mechanism of interleukin-10 gene induction in the Indian major carp, catla (Catla catla). Aquac Res 43:897–907
Rutledge RG, Cote C (2003) Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res 31(16):e93
Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59
Small BC, Murdock CA, Bilodeau-Bourgeois AL, Peterson BC, Waldbieser GC (2008) Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comp Biochem Physiol Part B 151:296–304
Liu C, Xin N, Zhai Y, Jiang L, Zhai J, Zhang Q, Qi J (2014) Reference gene selection for quantitative real-time RT-PCR normalization in the half-smooth tongue sole (Cynoglossus semilaevis) at different DEVELOPMENTAL stages, in various tissue types and on exposure to chemicals. PLoS ONE 9(3):e91715
Du Y, Zhang L, Xu F, Huang B, Zhang G, Li L (2013) Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR. Fish Shellfish Immunol 34(3):939–945
Li Z, Yang L, Wang J, Shi W, Pawar RA, Liu Y, Xu C, Cong W, Hu Q, Lu T, Xia F, Guo W, Zhao M, Zhang Y (2010) Beta-Actin is a useful internal control for tissue specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum. Fish Shellfish Immunol 29:89–93
Dhar AK, Bowers RM, Licon KS, Veazey G, Read B (2009) Validation of reference genes for quantitative measurement of immune gene expression in shrimp. Mol Immunol 46:1688–1695
Su J, Zhang R, Dong J, Yang C (2011) Evaluation of internal control genes for qRT-PCR normalization in tissues and cell culture for antiviral studies of grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 30(3):830–835
McCurley AT, Callard GV (2008) Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol 9:102
Deloffre LA, Andrade A, Filipe AI, Canario AV (2012) Reference genes to quantify gene expression during oogenesis in a teleost fish. Gene 506(1):69–75
Olsvik PA, Lie KK, Jordal AE, Nilsen TO, Hordvik I (2005) Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol 6:21
Fernandes JM, Mommens M, Hagen Ø, Babiak I, Solberg C (2008) Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol Part B 150(1):23–32
Mitter K, Kotoulas G, Magoulas A, Mulero V, Sepulcre P, Figueras A, Novoa B, Sarropoulou E (2009) Evaluation of candidate reference genes for qPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax). Comp Biochem Physiol Part B 153:340–347
Casadei R, Pelleri MC, Vitale L, Facchin F, Lenzi L, Canaider S, Frabetti F (2011) Identification of housekeeping genes suitable for gene expression analysis in the zebrafish. Gene Expr Patterns 11(3):271–276
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Authors are thankful to Director, Central Institute of Fisheries Education, Mumbai, India for providing all the facilities for the present work. The authors declare that they have no conflict of interest.
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Kumari, K., Pathakota, GB., Annam, PK. et al. Characterisation and Validation of House Keeping Gene for Expression Analysis in Catla catla (Hamilton). Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 85, 993–1000 (2015). https://doi.org/10.1007/s40011-014-0482-9
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DOI: https://doi.org/10.1007/s40011-014-0482-9