Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV |
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Authors: | Hua Yue Xiao-wen Lei Fa-long Yang Ming-Yi Li Cheng Tang |
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Institution: | 1.College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China2.China Animal Health and Epidemiology Center, Qingdao 266032, China |
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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. |
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Keywords: | Reference gene Chicken embryo fibroblast H5N1 avian influenza virus (AIV) Real-time PCR (RT-PCR) |
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