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

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 (2012) 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells. Virology Journal, 9 (230). ISSN 1743-422X

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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


Item Type: Article
Additional Information: Copy of Creative Commons Attribution License must accompany any deposit
Schools/Departments: University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Identification Number: https://doi.org/10.1186/1743-422X-9-230
Depositing User: de Sousa, Mrs Shona
Date Deposited: 01 Apr 2014 10:36
Last Modified: 16 Sep 2016 05:09
URI: http://eprints.nottingham.ac.uk/id/eprint/2790

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