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

Autor(es)

Silveira, Gilbert O. ; Amaral, Murilo S. ; Coelho, Helena S. ; Maciel, Lucas F. ; Pereira, Adriana S. A. ; Olberg, Giovanna G. O. ; Miyasato, Patricia A. ; Nakano, Eliana ; Verjovski-Almeida, Sergio

• Tipo de documento: Artigo Científico
• Fonte: SCIENTIFIC REPORTS; v. 11, n. 1, p. 18-pg., 2021-08-19.

Resumo

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. (AU)

• Processo FAPESP: 18/19591-2 - Anotação de RNAs longos não-codificantes e identificação de potenciais alvos terapêuticos em Schistosoma mansoni
• Beneficiário: Lucas Ferreira Maciel
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 16/10046-6 - Efeito de GSK343, um inibidor da histona metiltransferase EZH2, sobre o parasita Schistosoma mansoni
• Beneficiário: Adriana Silva Andrade Pereira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 18/24015-0 - Caracterização funcional de RNAs longos não-codificadores de proteínas em Schistosoma mansoni
• Beneficiário: Gilbert de Oliveira Silveira
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 18/23693-5 - Mecanismos de ação de RNAs longos não-codificadores envolvidos nos programas de ativação gênica em eucariotos
• Beneficiário: Sergio Verjovski Almeida
• Modalidade de apoio: Auxílio à Pesquisa - Temático