Computational protocol: Identification of reference genes for qPCR analysis during hASC long culture maintenance

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Protocol publication

[…] qPCR was performed using a CFX96 Connect apparatus (BioRad). The reactions were carried out in triplicates using the SYBR Green PCR super-mix (BioRad), following the manufacturer's instructions. Each reaction was performed on 5 ng of cDNA in a final volume of 10 μL, primers were used at a concentration of 300 nM. Thermocycler program consisted of an initial hot start cycle at 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec. To confirm product specificity, a melting curve analysis was performed after each amplification.The initial panel of genes was analyzed with custom plates preloaded with primers of interest (BioRad). For subsequent experiments, the primer sequences, reported in , were designed using the software Beacon Designer 7® (BioRad). [...] To define if the panel of RGs was stable, we used the algorithms NormFinder [], Best Keeper [] and IdealRef (a personal version of GeNorm) based on the algorithm published by Vandesompele et al. in 2002 []. Furthermore, in order to verify IdealRef reliability, results were compared to those obtained by the free software RefFinder []. [...] The Excel-based tool BestKeeper was developed by Pfaffl and colleagues and it is based on the concept that more stable is the gene expression lower is its Cq variation if the cDNA amount is constant. Cq values are used as input data then the algorithm calculates geometric (Geo Mean), arithmetic mean (Ar Mean) and different values expressing data variation. These values are: minimum and maximum Cq, Cq standard deviation (Std Dev), coefficient of variation (CV); furthermore, the ratio, expressed as x-fold, between minimum and maximum Cq related to the geometric mean and its standard deviation are also provided. Authors listed the genes considering Std Dev and CV and suggested that Cq values with Std Dev higher than 1 are considered inconsistent. They also suggested to use more than one RG in order to get more reliable results []. Moreover, the algorithm performs pair-wise correlation analyses to estimate inter-gene relations of all possible RG pairs. For every calculated correlation, the Pearson correlation coefficient (r) and the probability p value are calculated. All those highly correlated House Keeping Genes (HKGs) are combined into an index, and then the correlation between each candidate HKG and the index is described by the Pearson correlation coefficient (r), coefficient of determination (r2) and the p-value []. […]

Pipeline specifications

Software tools Beacon Designer, NormFinder, BestKeeper
Application qPCR
Organisms Homo sapiens