Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR

Abstract Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic stud...

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Autores principales:	Gilbert O. Silveira, Murilo S. Amaral, Helena S. Coelho, Lucas F. Maciel, Adriana S. A. Pereira, Giovanna G. O. Olberg, Patricia A. Miyasato, Eliana Nakano, Sergio Verjovski-Almeida
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e3e22ccbe69e4880829662ea5ea2957f
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spelling	oai:doaj.org-article:e3e22ccbe69e4880829662ea5ea2957f2021-12-02T15:10:39ZAssessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR10.1038/s41598-021-96055-72045-2322https://doaj.org/article/e3e22ccbe69e4880829662ea5ea2957f2021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-96055-7https://doaj.org/toc/2045-2322Abstract Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.Gilbert O. SilveiraMurilo S. AmaralHelena S. CoelhoLucas F. MacielAdriana S. A. PereiraGiovanna G. O. OlbergPatricia A. MiyasatoEliana NakanoSergio Verjovski-AlmeidaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-18 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gilbert O. Silveira Murilo S. Amaral Helena S. Coelho Lucas F. Maciel Adriana S. A. Pereira Giovanna G. O. Olberg Patricia A. Miyasato Eliana Nakano Sergio Verjovski-Almeida Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
description	Abstract Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.
format	article
author	Gilbert O. Silveira Murilo S. Amaral Helena S. Coelho Lucas F. Maciel Adriana S. A. Pereira Giovanna G. O. Olberg Patricia A. Miyasato Eliana Nakano Sergio Verjovski-Almeida
author_facet	Gilbert O. Silveira Murilo S. Amaral Helena S. Coelho Lucas F. Maciel Adriana S. A. Pereira Giovanna G. O. Olberg Patricia A. Miyasato Eliana Nakano Sergio Verjovski-Almeida
author_sort	Gilbert O. Silveira
title	Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
title_short	Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
title_full	Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
title_fullStr	Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
title_full_unstemmed	Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR
title_sort	assessment of reference genes at six different developmental stages of schistosoma mansoni for quantitative rt-pcr
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e3e22ccbe69e4880829662ea5ea2957f
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Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR

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