RNA Extraction and RT-PCR Analysis

Frozen cell pellets in RNAiso Plus were homogenized in Trizol (Qiagen, 79306). RNA was purified by phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. RNA concentration was determined using NanoDrop 2000 (ThermoScientific, USA). The integrity and quality of ribosomal 28S and 18S were determined on the agarose gel. Any traces of genomic DNA were removed by DNase (Invitrogen, Am2238) treatment for 30 min at 37 °C followed by heat inactivation using 50 mM EDTA (Sigma, E5134). cDNA synthesis and real-time PCR were done as described previously^{27 (link)}. Relative quantitation of gene expression was done using β-actin as the endogenous reference gene for normalization. All primer sequences used for RT-PCR are listed in Supplementary Table 1.