The qPCR reaction was carried out with 5 μl of a ten-fold diluted cDNA in a final volume of 20 μl using 10 μl of SsoFast™ EvaGreen® Supermix, (BioRad). The amplification was performed in a CFX96 Touch instrument (BioRad, Hercules, CA), using the following thermal conditions: 98°C for 3 min, then 40 cycles of 98°C for 5 s and 61°C for 1 min. Amplification uniqueness was confirmed by sequencing.
The expression ratio of the gene of interest was normalized relative to the abundance of two reference genes (β-actin and GAPDH) [19 (link)–21 (link)] to adjust for unbalanced samples and corrected for coexpression in the qRT-PCR. Each reaction was run in triplicate and no-template controls (NTC) were included in each run. Normalization was performed using the ΔΔ Cq method [22 (link)] that uses geometric mean of Cq values. Technical replicates were averaged and only those samples with standard error lower than 0.2 Cq were maintained. Data analysis was carried out with Bio-Rad CFX Manager software (ver. 3.2.2) and GenEx (ver.6). All values are presented as means with a confidence interval of 99%.
The expression ratio of the gene of interest was normalized relative to the abundance of two reference genes (β-actin and GAPDH) [19 (link)–21 (link)] to adjust for unbalanced samples and corrected for coexpression in the qRT-PCR. Each reaction was run in triplicate and no-template controls (NTC) were included in each run. Normalization was performed using the ΔΔ Cq method [22 (link)] that uses geometric mean of Cq values. Technical replicates were averaged and only those samples with standard error lower than 0.2 Cq were maintained. Data analysis was carried out with Bio-Rad CFX Manager software (ver. 3.2.2) and GenEx (ver.6). All values are presented as means with a confidence interval of 99%.
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