Selection and Stability Validation of Reference Gene Candidates for Transcriptional Analysis in Rousettus Aegyptiacus : [Preprint]

Bats are the only mammals capable of powered flight and their body temperature can reach up to 42°C during flight. Additionally, bats display robust type I IFN interferon (IFN-I) responses and some species constitutively express IFN-α. Reference genes with stable expression under temperature oscillations and IFN-I release are therefore critical for normalization of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data in bats. The expression stability of reference genes in Rousettus aegyptiacus remains elusive, although this species is frequently used in the infection research. We selected ACTB , EEF1A1 , GAPDH and PGK1 as candidate reference genes and evaluated their expression stability in various tissues and cells from this model bat species upon IFN-I treatment at 37°C and 40°C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest stability under all tested conditions. ACTB and GAPDH displayed unstable expression at 40°C and upon IFN-I treatment, respectively. By normalizing to EEF1A1 , we uncovered that GAPDH expression was significantly induced by IFN‑I in R. aegyptiacus . Our study identifies EEF1A1 as the most suitable reference gene for qRT-PCR studies and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and even bear relevance for non-volant mammals that show physiological fluctuations of core body temperature.