Computational protocol: Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment

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Protocol publication

[…] Primers and probes were designed in our laboratory using the primer analysis software Primer Express 3.0 (Applied Biosystems). TaqMan probes were synthesized by Applied Biosystems, and primers were synthesized by Invitrogen/Life Technologies. The primers and TaqMan probes used in the current study were selected to bind specifically to the cDNAs of Cg84u and other C. glabrata strains (Table ). The sequences of TaqMan probes and forward and reverse primers, the gene numbers, and the localization for each PCR assay for the 16 reference genes and 10 target genes assessed in this study are listed in Additional file , Additional file , and Additional file . [...] Non-normalized gene expression levels from our experimental data were analyzed to evaluate the expression stability of potential reference genes, using four different software programs: hkgFinder [], geNorm [,], BestKeeper [], and NormFinder []. The hkgFinder software computes the pooled standard deviation (SD) of non-normalized expression data from both phenotypes (i.e., azole-treated and untreated C. glabrata cells), the fold change (FC) values between the two phenotypes, and Student’s t-tests of the log2 fold-change values with Holm-adjusted P-values. The reference genes with the smallest SD and the smallest, non-significant FC are identified as the best potential reference genes (Additional file ). The geNorm software computes a stability value (M) and a pairwise variation (V), which are used to evaluate each individual reference gene candidate or each combination of reference genes, called a normalization factor (NF). The pairwise variability V of two genes j and k is the standard deviation of all log2 ratios of aj/ak, while the stability value M of gene j is the mean of all possible pairwise variations Vjk. Graphs of the M values help identify the best individual reference genes, and graphs of the V values identify the optimal number of reference genes for an NF. Note that an earlier version called geNorm Excel was produced as an add-in for MS Excel and it required several hand calculations to convert crossing point (CP) values into relative expression values. That version is now unavailable and the new geNorm PLUS from Biogazelle does not require those hand calculations. The BestKeeper software uses pairwise correlation to determine whether potential reference genes should be included in a BestKeeper Index, which is simply the geometric mean of the CP or cycle threshold (CT) values. The NormFinder software computes a different type of stability value (ρig) based on the intragroup and intergroup variation of the expression data. The software instructions from each package were followed when inputting the RT-qPCR data, fetching the output, and interpreting the analysis results. […]

Pipeline specifications

Software tools Primer Express, BestKeeper, NormFinder
Application qPCR
Organisms [Candida] glabrata
Chemicals Fluconazole