Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

• Published in: Scientific reports Vol. 13; no. 1; pp. 19384 - 15
• Main Authors: Davies, Katherine A., Welch, Stephen R., Sorvillo, Teresa E., Coleman-McCray, JoAnn D., Martin, María Laura, Brignone, Julia M., Montgomery, Joel M., Spiropoulou, Christina F., Spengler, Jessica R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.11.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/767/1424; 631/326/596/2555; Animal models; Animal tissues; Crimean hemorrhagic fever; Gene expression; Hemorrhage; Humanities and Social Sciences; Infectious diseases; Kidneys; Laboratory animals; Laboratory tests; multidisciplinary; Rodents; Science; Science (multidisciplinary); Tissue analysis
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-45740-w

Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean–Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species ( Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.