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.

Epigenetics regulates transcription and pathogenesis in the parasiteTrichomonas vaginalis

“…Using data from the exponential phase of the qPCR, threshold cycle base lines were set according to manufacturer protocols. Data from different samples were interpolated from standard curves established for each primer set and then normalized against the housekeeping alpha tubulin gene (dos Santos, de Vargas Rigo, Frasson, Macedo, & Tasca, ). The expression of housekeeping gene does not seem to change upon TSA treatment (data not shown).…”

A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis

“…To assess the expression levels of all 10 hydrogenase paralogs present in the T. vaginalis genome, we normalized their expression to the housekeeping gene of topoisomerase 2 43 (Figure S3). Four genes (TVAG_452140, TVAG_136330, TVAG_483690, and TVAG_447160) encode putatively cytosolic proteins (not found in the hydrogenosomal proteome 42 and without predicted organellar targeting peptide; Figure S2).…”

Genotyping of Trichomonas vaginalis isolates from Egypt

“…The failure of amplification of the three housekeeping genes at all loci for all isolates may be due a problem in the sensitivity of the PCR or due to the nature of the genes being single-copy genes, although these genes were previously selected [28] based on being readily amplified and sequenced on both DNA strands of all T. vaginalis isolates. Dos Santos et al [49] found variable stability in the expression of nine housekeeping reference genes under different culture conditions. Genes encoding α-tubulin, actin and DNA TopII were the most stable and conversely, the widely used T. vaginalis reference genes encoding GAPDH and β-tubulin were less stable [49] .…”

“…Dos Santos et al [49] found variable stability in the expression of nine housekeeping reference genes under different culture conditions. Genes encoding α-tubulin, actin and DNA TopII were the most stable and conversely, the widely used T. vaginalis reference genes encoding GAPDH and β-tubulin were less stable [49] .…”