title: Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.
abstract: Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Odelta
authors.initials: O
authors.lastname: dos Santos
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Graziela
authors.initials: G
authors.lastname: de Vargas Rigo
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Amanda Piccoli
authors.initials: AP
authors.lastname: Frasson
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Alexandre José
authors.initials: AJ
authors.lastname: Macedo
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Tiana
authors.initials: T
authors.lastname: Tasca
mesh_terms: D000199:Actins / Q000235:genetics / Q000592:standards; D000465:Algorithms; D004257:DNA Polymerase II / Q000235:genetics / Q000592:standards; D005296:Ferrous Compounds / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D059467:Transcriptome; D014246:Trichomonas vaginalis / Q000235:genetics* / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000592:standards
chemical_list: D000199:Actins; D005296:Ferrous Compounds; D000644:Quaternary Ammonium Compounds; D014404:Tubulin; C038178:ammonium ferrous sulfate; D004257:DNA Polymerase II