title: 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.
abstract: 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.
authors.affiliation: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK. suresh.kuchipudi@nottingham.ac.uk
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Kuchipudi
authors.forename: Meenu
authors.initials: M
authors.lastname: Tellabati
authors.forename: Rahul K
authors.initials: RK
authors.lastname: Nelli
authors.forename: Gavin A
authors.initials: GA
authors.lastname: White
authors.forename: Belinda Baquero
authors.initials: BB
authors.lastname: Perez
authors.forename: Sujith
authors.initials: S
authors.lastname: Sebastian
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Slomka
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Brookes
authors.forename: Ian H
authors.initials: IH
authors.lastname: Brown
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Dunham
authors.forename: Kin-Chow
authors.initials: KC
authors.lastname: Chang
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D004285:Dogs; D004372:Ducks; D020869:Gene Expression Profiling / Q000592:standards; D020043:Genes, Essential / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics*; D008168:Lung / Q000166:cytology / Q000821:virology; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D020545:Respiratory Mucosa / Q000166:cytology / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D013552:Swine
chemical_list: D000199:Actins; D012337:RNA, Ribosomal, 18S; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases