18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells
Identifieur interne : 000B96 ( Main/Exploration ); précédent : 000B95; suivant : 000B9718S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells
Auteurs : Suresh V. Kuchipudi [Royaume-Uni] ; Meenu Tellabati [Royaume-Uni] ; Rahul K. Nelli [Royaume-Uni] ; Gavin A. White [Royaume-Uni] ; Belinda Baquero Perez [Royaume-Uni] ; Sujith Sebastian [Royaume-Uni] ; Marek J. Slomka [Royaume-Uni] ; Sharon M. Brookes [Royaume-Uni] ; Ian H. Brown [Royaume-Uni] ; Stephen P. Dunham [Royaume-Uni] ; Kin-Chow Chang [Royaume-Uni]Source :
- Virology Journal [ 1743-422X ] ; 2012.
Abstract
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 (
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
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
Url:
DOI: 10.1186/1743-422X-9-230
PubMed: 23043930
PubMed Central: 3499178
Affiliations:
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Slomka</name><affiliation wicri:level="1"><nlm:aff id="I3">Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone</wicri:regionArea><wicri:noRegion>Addlestone</wicri:noRegion></affiliation></author><author><name sortKey="Brookes, Sharon M" sort="Brookes, Sharon M" uniqKey="Brookes S" first="Sharon M" last="Brookes">Sharon M. Brookes</name><affiliation wicri:level="1"><nlm:aff id="I3">Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone</wicri:regionArea><wicri:noRegion>Addlestone</wicri:noRegion></affiliation></author><author><name sortKey="Brown, Ian H" sort="Brown, Ian H" uniqKey="Brown I" first="Ian H" last="Brown">Ian H. 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Dunham</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Chang, Kin Chow" sort="Chang, Kin Chow" uniqKey="Chang K" first="Kin-Chow" last="Chang">Kin-Chow Chang</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23043930</idno><idno type="pmc">3499178</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499178</idno><idno type="RBID">PMC:3499178</idno><idno type="doi">10.1186/1743-422X-9-230</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000406</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000406</idno><idno type="wicri:Area/Pmc/Curation">000406</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000406</idno><idno type="wicri:Area/Pmc/Checkpoint">000532</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000532</idno><idno type="wicri:Area/Ncbi/Merge">000296</idno><idno type="wicri:Area/Ncbi/Curation">000296</idno><idno type="wicri:Area/Ncbi/Checkpoint">000296</idno><idno type="wicri:Area/Main/Merge">000B98</idno><idno type="wicri:Area/Main/Curation">000B96</idno><idno type="wicri:Area/Main/Exploration">000B96</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main"><italic>18S rRNA</italic> is a reliable normalisation gene for real time PCR based on influenza virus infected cells</title><author><name sortKey="Kuchipudi, Suresh V" sort="Kuchipudi, Suresh V" uniqKey="Kuchipudi S" first="Suresh V" last="Kuchipudi">Suresh V. Kuchipudi</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Tellabati, Meenu" sort="Tellabati, Meenu" uniqKey="Tellabati M" first="Meenu" last="Tellabati">Meenu Tellabati</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Nelli, Rahul K" sort="Nelli, Rahul K" uniqKey="Nelli R" first="Rahul K" last="Nelli">Rahul K. Nelli</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="White, Gavin A" sort="White, Gavin A" uniqKey="White G" first="Gavin A" last="White">Gavin A. White</name><affiliation wicri:level="4"><nlm:aff id="I2">School of Biosciences, University of Nottingham, Sutton Bonington Campus, College Road,, Loughborough, Leicestershire, LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Biosciences, University of Nottingham, Sutton Bonington Campus, College Road,, Loughborough, Leicestershire, LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Perez, Belinda Baquero" sort="Perez, Belinda Baquero" uniqKey="Perez B" first="Belinda Baquero" last="Perez">Belinda Baquero Perez</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Sebastian, Sujith" sort="Sebastian, Sujith" uniqKey="Sebastian S" first="Sujith" last="Sebastian">Sujith Sebastian</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Slomka, Marek J" sort="Slomka, Marek J" uniqKey="Slomka M" first="Marek J" last="Slomka">Marek J. Slomka</name><affiliation wicri:level="1"><nlm:aff id="I3">Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone</wicri:regionArea><wicri:noRegion>Addlestone</wicri:noRegion></affiliation></author><author><name sortKey="Brookes, Sharon M" sort="Brookes, Sharon M" uniqKey="Brookes S" first="Sharon M" last="Brookes">Sharon M. Brookes</name><affiliation wicri:level="1"><nlm:aff id="I3">Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone</wicri:regionArea><wicri:noRegion>Addlestone</wicri:noRegion></affiliation></author><author><name sortKey="Brown, Ian H" sort="Brown, Ian H" uniqKey="Brown I" first="Ian H" last="Brown">Ian H. Brown</name><affiliation wicri:level="1"><nlm:aff id="I3">Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Virology department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone</wicri:regionArea><wicri:noRegion>Addlestone</wicri:noRegion></affiliation></author><author><name sortKey="Dunham, Stephen P" sort="Dunham, Stephen P" uniqKey="Dunham S" first="Stephen P" last="Dunham">Stephen P. Dunham</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author><author><name sortKey="Chang, Kin Chow" sort="Chang, Kin Chow" uniqKey="Chang K" first="Kin-Chow" last="Chang">Kin-Chow Chang</name><affiliation wicri:level="4"><nlm:aff id="I1">School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough, Leicestershire LE12 5RD</wicri:regionArea><orgName type="university">Université de Nottingham</orgName><placeName><settlement type="city">Nottingham</settlement><region type="nation">Angleterre</region><region type="région" nuts="1">Nottinghamshire</region></placeName></affiliation></author></analytic><series><title level="j">Virology Journal</title><idno type="eISSN">1743-422X</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>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 (<italic>ACTB</italic>) and glyceraldehyde-3-phosphate dehydrogenase (<italic>GAPDH</italic>), under different experimental settings. However, <italic>ACTB</italic> and <italic>GAPDH</italic> 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 [<italic>ACTB</italic>, <italic>GAPDH</italic>, 18S ribosomal RNA (<italic>18S rRNA</italic>), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (<italic>ATP5B</italic>) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (<italic>ATP5G1</italic>)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.</p></sec><sec><title>Results</title><p>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 <italic>NormFinder</italic> and <italic>BestKeeper</italic> software programmes found that <italic>18S rRNA</italic> was the most stable gene in HBECs, PTECs and avian lung cells.</p></sec><sec><title>Conclusions</title><p>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 <italic>18S rRNA</italic> is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including <italic>ACTB</italic> and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Huggett, J" uniqKey="Huggett J">J Huggett</name></author><author><name sortKey="Dheda, K" uniqKey="Dheda K">K Dheda</name></author><author><name sortKey="Bustin, S" uniqKey="Bustin S">S Bustin</name></author><author><name sortKey="Zumla, A" uniqKey="Zumla A">A Zumla</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, Sa" uniqKey="Bustin S">SA Bustin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mccurley, A" uniqKey="Mccurley A">A McCurley</name></author><author><name sortKey="Callard, G" uniqKey="Callard G">G 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Kuchipudi</name></region><name sortKey="Brookes, Sharon M" sort="Brookes, Sharon M" uniqKey="Brookes S" first="Sharon M" last="Brookes">Sharon M. Brookes</name><name sortKey="Brown, Ian H" sort="Brown, Ian H" uniqKey="Brown I" first="Ian H" last="Brown">Ian H. Brown</name><name sortKey="Chang, Kin Chow" sort="Chang, Kin Chow" uniqKey="Chang K" first="Kin-Chow" last="Chang">Kin-Chow Chang</name><name sortKey="Dunham, Stephen P" sort="Dunham, Stephen P" uniqKey="Dunham S" first="Stephen P" last="Dunham">Stephen P. Dunham</name><name sortKey="Nelli, Rahul K" sort="Nelli, Rahul K" uniqKey="Nelli R" first="Rahul K" last="Nelli">Rahul K. Nelli</name><name sortKey="Perez, Belinda Baquero" sort="Perez, Belinda Baquero" uniqKey="Perez B" first="Belinda Baquero" last="Perez">Belinda Baquero Perez</name><name sortKey="Sebastian, Sujith" sort="Sebastian, Sujith" uniqKey="Sebastian S" first="Sujith" last="Sebastian">Sujith Sebastian</name><name sortKey="Slomka, Marek J" sort="Slomka, Marek J" uniqKey="Slomka M" first="Marek J" last="Slomka">Marek J. Slomka</name><name sortKey="Tellabati, Meenu" sort="Tellabati, Meenu" uniqKey="Tellabati M" first="Meenu" last="Tellabati">Meenu Tellabati</name><name sortKey="White, Gavin A" sort="White, Gavin A" uniqKey="White G" first="Gavin A" last="White">Gavin A. White</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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