| Full text | |
| Author(s): |
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
Total Authors: 9
|
| Document type: | Journal article |
| Source: | SCIENTIFIC REPORTS; v. 11, n. 1, p. 18-pg., 2021-08-19. |
| 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. (AU) | |
| FAPESP's process: | 18/19591-2 - Long non-coding RNAs annotation and identification of potential therapeutic targets in Schistosoma mansoni |
| Grantee: | Lucas Ferreira Maciel |
| Support Opportunities: | Scholarships in Brazil - Master |
| FAPESP's process: | 16/10046-6 - Effect of GSK343, an inhibitor of the histone methyltransferase EZH2, on the parasite Schistosoma mansoni |
| Grantee: | Adriana Silva Andrade Pereira |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 18/24015-0 - Functional characterization of long non-protein coding RNAs in Schistosoma mansoni |
| Grantee: | Gilbert de Oliveira Silveira |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 18/23693-5 - Mechanisms of action of long non-coding RNAs involved with gene activation programs in eukaryotes |
| Grantee: | Sergio Verjovski Almeida |
| Support Opportunities: | Research Projects - Thematic Grants |