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

Abstract 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 temperatu...

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Autores principales:	Virginia Friedrichs, Anne Balkema-Buschmann, Anca Dorhoi, Gang Pei
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5944f24a6a3741b8a745c24799be642e
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spelling	oai:doaj.org-article:5944f24a6a3741b8a745c24799be642e2021-11-08T10:51:23ZSelection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus10.1038/s41598-021-01260-z2045-2322https://doaj.org/article/5944f24a6a3741b8a745c24799be642e2021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01260-zhttps://doaj.org/toc/2045-2322Abstract 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.Virginia FriedrichsAnne Balkema-BuschmannAnca DorhoiGang PeiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Virginia Friedrichs Anne Balkema-Buschmann Anca Dorhoi Gang Pei Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
description	Abstract 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.
format	article
author	Virginia Friedrichs Anne Balkema-Buschmann Anca Dorhoi Gang Pei
author_facet	Virginia Friedrichs Anne Balkema-Buschmann Anca Dorhoi Gang Pei
author_sort	Virginia Friedrichs
title	Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
title_short	Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
title_full	Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
title_fullStr	Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
title_full_unstemmed	Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus
title_sort	selection and stability validation of reference gene candidates for transcriptional analysis in rousettus aegyptiacus
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/5944f24a6a3741b8a745c24799be642e
work_keys_str_mv	AT virginiafriedrichs selectionandstabilityvalidationofreferencegenecandidatesfortranscriptionalanalysisinrousettusaegyptiacus AT annebalkemabuschmann selectionandstabilityvalidationofreferencegenecandidatesfortranscriptionalanalysisinrousettusaegyptiacus AT ancadorhoi selectionandstabilityvalidationofreferencegenecandidatesfortranscriptionalanalysisinrousettusaegyptiacus AT gangpei selectionandstabilityvalidationofreferencegenecandidatesfortranscriptionalanalysisinrousettusaegyptiacus
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Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus

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