| Author | Brito, Márcio Wilson Dias de | |
| Author | Carvalho, Stephanie Serafim de | |
| Author | Mota, Maria Beatriz dos Santos | |
| Author | Mesquita, Rafael Dias | |
| Access date | 2024-07-23T18:42:18Z | |
| Available date | 2024-07-23T18:42:18Z | |
| Document date | 2024 | |
| Citation | BRITO, Márcio Wilson Dias de et al. RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR. BMC Genomics, v. 25, n. 697, p. 1-12, 16 July 2024. | |
| ISSN | 1471-2164 | |
| URI | https://www.arca.fiocruz.br/handle/icict/65120 | |
| Sponsorship | Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ): grant number E-26/260.004/2021 (Márcio Wilson Dias de Brito and Maria Beatriz dos Santos Mota). | |
| Sponsorship | Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); grant number: 465678/2014-9 (Rafael Dias Mesquita). | |
| Language | eng | en_US |
| Publisher | BMC | |
| Rights | open access | |
| Title | RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR | en_US |
| Type | Article | |
| DOI | 10.1186/s12864-024-10511-y | |
| Abstract | Background: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. Results: We developed a software named “Gene Selector for Validation” (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. Conclusion: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data. | en_US |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Programa de Pós-Graduação em Biologia Computacional e Sistemas. Rio de Janeiro, RJ, Brasil / RioGen Tecnologia. Rio de Janeiro, RJ, Brasil. | |
| Affilliation | Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil. | |
| Affilliation | RioGen Tecnologia. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Centro de Ciências Matemáticas e da Natureza. Instituto de Química. Departamento de Bioquímica. Rio de Janeiro, RJ, Brasil. | |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Centro de Ciências Matemáticas e da Natureza. Instituto de Química. Departamento de Bioquímica. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica Leopoldo de Meis. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil. | |
| Subject | Gene expression | en_US |
| Subject | Reference genes | en_US |
| Subject | Transcriptome validation | en_US |
| Subject | Aedes aegypti | en_US |
| Subject | RNA-seq | en_US |
| Subject | RT-qPCR | en_US |
| e-ISSN | 1471-2164 | |
