Quantitative Real-Time PCR Protocol

cDNA was loaded into each well of a 96-well plate containing forward and reverse primers (primer sequences are given in supplemental online Table 2), 10 µl of 2× Power SYBR master mix (Thermo Fisher Scientific Life Sciences), and RNase-free H₂O to a final volume of 20 μl. The reaction was carried out in a Bio-Rad PTC-200 thermal cycler equipped with a Chromo4 real time detection system (Bio-Rad, Hercules, CA, http://www.bio-rad.com/), and the results were processed using Opticon Monitor 3 software (Bio-Rad). Each assay was performed in triplicate, averaged, and normalized to an endogenous control (β₂-microglobulin). Comparisons were made across experimental conditions using the ΔΔC_T method [44 (link), 45 (link)]. Data are displayed as the log₂ of the fold change in normalized expression. Primer sequences are included in supplemental online Table 1.