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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 35 °C, 37 °C and 40 °C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that  EEF1A1  exhibited the highest expression stability under all tested conditions.  ACTB  and  GAPDH  displayed unstable expression upon temperature change and 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 upon temperature changes and IFN-I treatment and unveils the induction of  GAPDH  expression by IFN-I in  R. aegyptiacus.  These findings are pertinent to other bat species and may be relevant for non-volant mammals that show physiological fluctuations of core body temperature.

Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus