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Background  One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin ( ACTB ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), under different experimental settings. However,  ACTB  and  GAPDH  remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ ACTB ,  GAPDH , 18S ribosomal RNA ( 18S rRNA ), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide ( ATP5B ) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) ( ATP5G1 )] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.    Results  The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using  NormFinder  and  BestKeeper  software programmes found that  18S rRNA  was the most stable gene in HBECs, PTECs and avian lung cells.    Conclusions  Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that  18S rRNA  is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including  ACTB  and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

18S rRNAis a reliable normalisation gene for real time PCR based on influenza virus infected cells