Abstract
Expression of four reference genes of grass carp, including β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1α), was studied in tissues of normal individuals and bacteria-infected individuals. EF1α had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1α in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1α is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andersen CL, Jensen JL, Ørntoft 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
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39
Chang MX, Sun BJ, Nie P (2007) The first non-mammalian CXCR3 in a teleost fish: gene and expression in blood cells and central nervous system in the grass carp (Ctenopharyngodon idella). Mol Immunol 44(6):1123–1134
Fernandes JMO, Mommens M, Hagen Ø et al (2008) Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B 150:23–32
Infante C, Matsuoka MP, Asensio E et al (2008) Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol 9:28
Ingerslev HC, Pettersen EF, Jakobsen RA et al (2006) Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol 43:1194–1201
Jorgensen SM, Kleveland EJ, Grimholt U et al (2006) Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Mar Biotechnol 8:398–408
Larsen AN, Solstad T, Svineng G et al (2009) Molecular characterisation of a goose-type lysozyme gene in Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol 26:122–132
Liu Y, Wei L, Li N et al (2008) Effect of formalin inactivated Flavobacterium columnare on expression of immune related genes in grass carp, Ctenopharyngodon idellus. Acta Hydrobiol Sin 32(6):794–801
Olsvik PA, Kai KL, Ann-Elise OJ et al (2005) Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol 6:21
Pfaffl MW, 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. Biotechnol Lett 26(6):509–515
Ruan W, Lai M (2007) Actina, a reliable marker of internal control? Clin Chim Acta 385:1–5
Small BC, Murdock CA, Bilodeau-Bourgeois L et al (2008) Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comp Biochem Physiol B 151:296–304
Tang R, Dodd A, Lai D et al (2007) Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin 39(5):384–390
Vandesompele J, De PK, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):0034.1–0034.11
Ye X, Zhang L, Tian Y, Tan A et al (2010) Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idellus. Dev Comp Immunol 34(5):501–509
Acknowledgements
This work was supported by grants from the Science and Technology Key Project in Agricultural Areas of Guangdong Province (No. 2008A020100016), Science and Technology Project of Ocean and Fisheries of Guangdong Province (No. A200899F01), Science and Technology Project of Guangzhou (No. 20084411115, N0.2009J1-C021), the earmarked fund for Modern Agro-indursty Technology Research System (Nycytx-49-07).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ye, X., Zhang, L., Dong, H. et al. Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR. Biotechnol Lett 32, 1031–1038 (2010). https://doi.org/10.1007/s10529-010-0258-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-010-0258-0