Abstract
Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Al-Azemi A, Bahl J, Al-Zenki S,et al. 2008. Avian influenza A virus (H5N1) outbreaks, Kuwait, 2007. Emerg Infect Dis, 14: 958–961.
Burnside J, Ouyang M, Anderson A,et al. 2008. Deep sequencing of chicken microRNAs. BMC Genomics, 22:185.
Bustin S A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mo Endocrinol, 25: 169–193.
de Kok J B, Roelofs R W, Giesendorf B A,et al. 2005. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest, 85: 154–159.
Fang L Q, de Vlas S J, Liang S,et al. 2008. Environmental factors contributing to the spread of H5N1 avian influenza in mainland China. PLoS One, 5: 2268.
Giulietti A, Overbergh L, Valckx D,et al. 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25: 386–401.
Glare E M, Divjak M, Bailey M J,et al. 2002. Beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax, 57: 765–770.
Haberhausen G, Pinsl J, Kuhn C C,et al. 1998. Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays. J Clin Microbiol, 36: 628–633.
Jang J, Hong S H, Choi D,et al. 2010. Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts. Appl Microbiol Biotechnol, 85: 1509–1520.
Jenkins K A, Bean A G, Lowenthal J W. 2007. Avian genomics and the innate immune response to viruses. Cytogenet Genome Res, 117: 207–212.
Li Y P, Band D D, Handberg K J,et al. 2005. Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus. Vet Microbiol, 110: 155–165.
Mackay I M, Arden K E, Nitsche A. 2002. Real-time PCR in virology. Nucl Acids Res, 30: 1292–1305.
Pfaffl M W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res, 29: 45.
Radonić A, Thulke S, Bae H G,et al. 2005. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections. Virology J, 2: 7.
Sarmento L, Afonso C L, Estevez C,et al. 2008. Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses.Vet Immunol Immunopathol, 125: 291–302.
Selvey S, Thompson E W, Matthaei K,et al. 2001. Beta- actin-an unsuitable internal control for RT-PCR. Mol Cell Probes, 15: 307–311.
Shi L, Li H, Ma G,et al. 2009.Competitive replication of different genotypes of infectious bursal disease virus on chicken embryo fibroblasts. Virus Genes, 39: 46–52.
Shirin M, Jagdev M. Sharm A,et al. 2005. Transcriptional response of avian cellsto infection with Newcastle disease virus. Virus Res, 107: 103–108.
Suzuki T, Higgins P J, Crawford D R. 2000. Control selection for RNA quantitation. Biotechniques, 29: 332–337.
Thellin O, Zorzi W, Lakaye B,et al. 1999. Housekeeping genes as internal standards: use and limits. J Biotechnol, 75: 291–295.
Vandesompele J, De Preter K, 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:research00343.
Warrington J A, Nair A, Mahadevappa M,et al. 2000. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics, 2: 143–147.
Watson S, Mercier S, Bye C,et al. 2007. Determination of suitable housekeeping genes for normalisation of quantitative real time PCR analysis of cells infected with human immunodeficiency virus and herpes viruses. Virol J, 4: 130.
Webster R G, Peiris M, Chen H,et al. 2006. H5N1 outbreaks and enzootic influenza. Emerg Infect Dis, 12: 3–8.
Wong S Y, Yuen K. 2006. Avian influenza virus infections in humans. Chest, 129: 156–168.
Xu H, Yao Y, Zhao Y,et al. 2008. Analysis of the expression profiles of Marek’s disease virus-encoded microRNAs by real-time quantitative PCR. J Virol Methods, 149: 201–208.
Zheng Y, Liu J H. 1997. In: Animal virology, 2nd ed. Beijing, Science Press.
Zhong H, Simons J W. 1999. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem Biophys Res Commun, 259: 523–526.
Author information
Authors and Affiliations
Corresponding author
Additional information
Foundation item: National “11th Five-year Plan” Scientific and Technical Supporting Programs (2006BAD06A11).
Rights and permissions
About this article
Cite this article
Yue, H., Lei, Xw., Yang, Fl. et al. Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV. Virol. Sin. 25, 425–431 (2010). https://doi.org/10.1007/s12250-010-3114-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12250-010-3114-4