Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro
Identifieur interne : 000804 ( Main/Exploration ); précédent : 000803; suivant : 000805Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro
Auteurs : Marion Griessl [États-Unis] ; Michael Gutknecht [États-Unis] ; Charles H. Cook [États-Unis]Source :
- Journal of virological methods [ 0166-0934 ] ; 2017.
Abstract
Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for
Url:
DOI: 10.1016/j.jviromet.2017.06.012
PubMed: 28655566
PubMed Central: 6775634
Affiliations:
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Cook</name><affiliation wicri:level="2"><nlm:aff id="A1">Department of Surgery, Division of Acute Care Surgery, Trauma and Surgical Critical Care, Beth Israel Deaconess Medical Center - Harvard Medical School, Boston, MA</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Massachusetts</region></placeName><wicri:cityArea>Department of Surgery, Division of Acute Care Surgery, Trauma and Surgical Critical Care, Beth Israel Deaconess Medical Center - Harvard Medical School, Boston</wicri:cityArea></affiliation></author></analytic><series><title level="j">Journal of virological methods</title><idno type="ISSN">0166-0934</idno><idno type="eISSN">1879-0984</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P2">Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for <italic>in vitro</italic> infection studies. For <italic>in vivo</italic> studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.</p></div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Massachusetts</li></region></list><tree><country name="États-Unis"><region name="Massachusetts"><name sortKey="Griessl, Marion" sort="Griessl, Marion" uniqKey="Griessl M" first="Marion" last="Griessl">Marion Griessl</name></region><name sortKey="Cook, Charles H" sort="Cook, Charles H" uniqKey="Cook C" first="Charles H" last="Cook">Charles H. Cook</name><name sortKey="Gutknecht, Michael" sort="Gutknecht, Michael" uniqKey="Gutknecht M" first="Michael" last="Gutknecht">Michael Gutknecht</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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