Abstract
Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.
Highlights
Quantitative real-time PCR (QPCR) has become the favoured tool in mRNA expression analysis and in virus diagnostics [1]
The most prominent problem in quantitative mRNA expression analysis is the selection of an appropriate control gene
The glyceraldehyde 3-phosphate dehydrogenase (GAP) gene and the β-actin (Act) gene were used as control genes in classical molecular methods for RNA detection
Summary
Quantitative real-time PCR (QPCR) has become the favoured tool in mRNA expression analysis and in virus diagnostics [1]. Real-time PCR has outperformed classical and semi-quantitative PCR methods in terms of accuracy, reproducibility, safety and convenience for the precise monitoring of viral load in clinical material, as well as for the investigation of the expression of cellular genes in response to virus infection. The glyceraldehyde 3-phosphate dehydrogenase (GAP) gene and the β-actin (Act) gene were used as control genes in classical molecular methods for RNA detection. The selection of the 10 most promising reference genes, GAP, Act, peptidyl prolyl isomerase A (PPI), glucose 6-phosphate dehydrogenase (G6P), TATABox binding protein (TBP), β2-microglobulin (β2M), αtubulin (Tub), ribosomal protein L13 (L13), phospholipase A2 (PLA) and RNA polymerase II (RPII) were evaluated in cell lines infected with members of different virus families: coronavirus (SARS-coronavirus), flavivirus (yellow fever virus, (YF)), herpesvirus (Human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV)) and orthopoxvirus camelpox (CAMP), covering DNA and RNA viruses
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