Quantitative Real-Time RT-PCR

RNA was isolated using TRI Reagent (Sigma Aldrich T9424), digested using DNase I (Sigma Aldrich AMPD1) to remove contaminating DNA, then reverse transcribed using Tetro cDNA Synthesis kit (Bioline BIO-65042) according to manufacturer’s instructions. Real-time PCR analyses were performed on diluted cDNA using the SensiFAST SYBR No-ROX Kit (Bioline BIO-98002) after identifying the linear range for cDNA dilutions and assessing melt temperature and amplicon sizes.

Relative expression levels were calculated by the 2^-ΔΔC_T method using the cycle threshold (C_T) of the gene of interest and the housekeeping gene (RNA polymerase II) [49] obtained from Corbett Rotor-Gene 6000 Series Software 1.7.