Abstract
BackgroundThe selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J).FindingsThe expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF.ConclusionsThe RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.
Highlights
The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections
The RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during Avian leukosis virus subgroup J (ALV-J) infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes
To identify suitable reference genes for mRNA analysis in poultry, here we present the expression stability of 11 housekeeping genes in chicken embryonic fibroblasts (CEF) after ALV-J infection, and propose stably expressed genes for use as reference genes in ALV-J/CEF settings
Summary
The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J). To ensure a reliable result in gene expression analysis, the selection of stably expressed reference gene (or genes) is an important technical prerequisite for each individual experimental setting [6]. This is especially important because even the expression stability of candidate reference housekeeping genes varies across host tissue cells, and virus strains [7,8]. To identify suitable reference genes for mRNA analysis in poultry, here we present the expression stability of 11 housekeeping genes in CEF after ALV-J infection, and propose stably expressed genes for use as reference genes in ALV-J/CEF settings
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