Kuchipudi, Suresh V. and Tellabati, Meenu and Nelli, Rahul K. and White, Gavin A. and Baquero Perez, Belinda and Sebastian, Sujith and Slomka, Marek J. and Brookes, Sharon M. and Brown, Ian H. and Dunham, Stephen P. and Chang, Kin-Chow
18S rRNA is a reliable normalisation gene for real
time PCR based on influenza virus infected cells.
Virology Journal, 9
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
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