Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques

Abstract

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.

Decreased myoblast differentiation in chronic binge alcohol-administered simian immunodeficiency virus-infected male macaques: role of decreased miR-206

“…Target genes were compared with the endogenous control [ribosomal protein S13 (RPS13)]. We previously reported that RPS13 is a reliable reference gene for normalization of mRNA in tissues of SIV-infected macaques and does not change with different treatments and tissues (16,51,55). Results from this study confirmed that RPS13 did not significantly change with treatments.…”

Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR

“…RPS13 has been identified as the most stable gene in different tissues of Sesamia inferens 48 , and RPS13 mRNA expression was shown to be unaffected by alcohol administration, SIV infection, or antiviral therapy in rhesus macaques 49 . Our results demonstrated that RPS13 was the most stable gene both among different developmental stages and among different strains of C. ferrugineus , indicating that RPS13 exhibits a wide degree of stability in a range of conditions.…”

Lack of susceptibility in neonatally infected rhesus macaques to simian immunodeficiency virus-induced encephalitis

“…Brain associated viral load Quantitative real-time PCR (qPCR) assays were completed on fresh-frozen brain samples snap frozen in liquid nitrogen or OCT using previously published methodologies (Robichaux et al 2016;Lee et al 2016). Duplicates were run for sample reliability.…”

“…Duplicates were run for sample reliability. The limit of detection per 1,000,000 cells was 50 copies for SIV RNA and 10 copies for SIV DNA (Robichaux et al 2016;Lee et al 2016). For statistical comparisons, samples with undetectable levels of SIV were reported as 25 for SIV RNA and 5 for SIV DNA, and all values were log-10 transformed (Hornung and Reed 1990).…”