Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro
| Autor: | Marion Griessl, Michael Gutknecht, Charles H. Cook |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Hypoxanthine Phosphoribosyltransferase Muromegalovirus Cell type 030106 microbiology Gene Expression Biology Real-Time Polymerase Chain Reaction Article Mice 03 medical and health sciences In vivo Reference genes Virology Gene expression Animals Lung Gene Mice Inbred BALB C Glyceraldehyde-3-Phosphate Dehydrogenases Herpesviridae Infections Molecular biology In vitro Reverse transcriptase 030104 developmental biology Real-time polymerase chain reaction Liver NIH 3T3 Cells Algorithms |
| Zdroj: | Journal of Virological Methods |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2017.06.012 |
| Popis: | 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 in vitro infection studies. For in vivo 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. |
| Databáze: | OpenAIRE |
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