Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques
| Autor: | Nedra Lacour, Spencer Robichaux, Gregory J. Bagby, Angela M. Amedee |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Ribosomal Proteins
0301 basic medicine Real-Time Polymerase Chain Reaction Article 03 medical and health sciences 0302 clinical medicine Virology TaqMan Animals Messenger RNA biology Reverse Transcriptase Polymerase Chain Reaction Reproducibility of Results RNA Reference Standards Viral Load biology.organism_classification Macaca mulatta Molecular biology In vitro Reverse transcriptase Rhesus macaque 030104 developmental biology Real-time polymerase chain reaction Simian Immunodeficiency Virus Viral load 030217 neurology & neurosurgery |
| Zdroj: | Journal of Virological Methods. 236:245-251 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2016.08.004 |
| Popis: | Persistent HIV reservoirs and the absolute quantification of viral RNA copies in tissues have become a prominent focus of multiple areas of HIV/SIV research. Absolute quantification of viral RNA via reverse transcription, quantitative PCR (RT-qPCR) necessitates the use of an appropriate RNA reference gene whose expression is unaffected by both experimental and confounding conditions. In this study, we demonstrate the utility of ribosomal protein S13 mRNA (RPS13) as a stable, medium abundance reference gene for RT-qPCR normalization of HIV/SIV RNA copy number. We developed a RPS13 RNA standard assay utilizing an in vitro RNA transcript for normalization of absolute SIV RNA quantities in tissues reservoirs. The RT-qPCR assay showed a high degree of repeatability and reproducibility across RNA levels appropriate for absolute SIV quantification. In assessing the utility of RPS13 as a reference gene, limited variation in the absolute, inter-tissue quantities of RPS13 mRNA was observed within multiple tissue samples obtained from rhesus macaques (average CV = 2.86%). We demonstrate rhesus macaque RPS13 mRNA expression is not affected by alcohol administration, SIV infection, or antiviral therapy (PMPA/FTC). Additionally, assay functionality was validated for normalization of SIV copy number using cellular RNA prepared from samples of variable RNA integrity. RPS13 is a suitable reference gene for normalization of absolute SIV RNA quantities in tissues and is most appropriate for intra-tissue or similar tissue type comparisons of SIV copy number. |
| Databáze: | OpenAIRE |
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