Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of Spodoptera frugiperda Cells during AcMNPV Infection
Identifieur interne : 001136 ( Main/Exploration ); précédent : 001135; suivant : 001137Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of Spodoptera frugiperda Cells during AcMNPV Infection
Auteurs : Tamer Z. Salem [Égypte] ; Walaa R. Allam [Égypte] ; Suzanne M. Thiem [États-Unis]Source :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection.
Url:
DOI: 10.1371/journal.pone.0108516
PubMed: 25313905
PubMed Central: 4196776
Affiliations:
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Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Verifying the Stability of Selected Genes for Normalization in Q PCR Experiments of <italic>Spodoptera frugiperda</italic>
Cells during AcMNPV Infection</title>
<author><name sortKey="Salem, Tamer Z" sort="Salem, Tamer Z" uniqKey="Salem T" first="Tamer Z." last="Salem">Tamer Z. Salem</name>
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<author><name sortKey="Allam, Walaa R" sort="Allam, Walaa R" uniqKey="Allam W" first="Walaa R." last="Allam">Walaa R. Allam</name>
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<series><title level="j">PLoS ONE</title>
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<front><div type="abstract" xml:lang="en"><p>It is challenging to find genes with stable transcripts for use as reference genes for quantitative realtime polymerase chain reaction (qRT-PCR) during viral infection. <italic>Autographa californica</italic>
nucleopolyhedrovirus (AcMNPV) is known to globally shut off host gene transcription in Sf21 cells and to modify their cytoskeletons. In this study, seven host genes were selected for validation as references for gene expression experiments using qRT-PCR. Two of them, ecdysoneless (ECD) and myosin showed stable RNA levels in our previous microarray study at 6, 12, and 24 hpi for both genes and 48 hpi for ECD. The others, actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 28S ribosome (28S), are commonly employed as reference genes for qRT-PCR. Ribosomal protein L35 (L35) gene was selected to test if ribosomal protein genes show stable RNA transcript levels similar to 28S and 18S rRNA and to validate the microarray data. In addition to 28S, previously known to have stable transcript levels, qRT-PCR showed that ECD transcript levels remained constant throughout the time course of AcMNPV infection. Transcripts of cytoskeleton genes such as actin, tubulin, and myosin declined dramatically as the infection progressed. GAPDH and L35 transcripts also declined over time. These results indicate that ECD is a reliable reference gene for qRT-PCR experiments during AcMNPV infection of <italic>Spodoptera frugiperda</italic>
cells. Although 28S could be used as a reference gene for these experiments, it is less useful than ECD because of its abundance, which might make it difficult to establish an accurate baseline value for data analysis.</p>
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