Computational protocol: Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

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Protocol publication

[…] The Primer3 designing software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) was used to design primer pairs using the following criteria: product size between 100 and 200 bp, Tm of approximately 60°C, GC content of 40–60% and primers length of 18–22 bp. The generated primer pair for each gene was then aligned against the T. vaginalis genome to confirm its specificity in silico. The primer pairs were also evaluated for primer dimer formation using multiple primer analyzers (http://www.thermoscientificbio.com/webtools/multipleprimer/). The forward and reverse primers were intentionally targeted to the adjoining exons, which were separated by an intron. The gene ID, primer sequence and gene symbol are shown in for all nucleotide sequences assessed here. [...] To determine the best reference genes among the different culture conditions, two independent statistical algorithms were used, geNorm and NormFinder [,]. For the geNorm analysis, the raw data Ct values were entered into the geNorm of the qBasePLUS V 2.4 software [], and for the NormFinder, the raw data were transformed to the relative quantities using the delta-Ct method Q = 2-ΔCt []. NormFinder was used to calculate the stability value of the reference genes based on their intra- and inter-expression variation, and those that exhibited lower average expression stability values were regarded as more stably expressed reference genes. The geNorm software was used to calculate the expression stability value (M) and the mean pairwise variation (V value, V n/n+1) between all of the tested genes. The threshold of V < 0.15 was used in this study [,]. […]

Pipeline specifications

Software tools Primer3, NormFinder
Application qPCR
Organisms Trichomonas vaginalis
Diseases Prostatic Neoplasms, Trichomonas Infections, HIV Infections