Quantifying Gene Expression in Cells

mRNA was isolated using an mRNA extraction kit according to the manufacturer’s instructions (Qiagen, Germantown, MD, USA). RNA quality and quantity were assessed NanoDrop Spectrophotometer at 260 nm and 280 nm absorbance. cDNA was generated and the following genes were studied (corresponding primer sequences given in references in parentheses): Wnt-11, NSE, Hes6, Neuro D [16 (link)]; Nanog [30 (link)]; Vim, Snail, Twist, E-cadherin (CDH1) [31 (link)]. qRT-PCR analysis was done using Taq SYBR Green premix (Qiagen, Germantown, MD, USA) and the following conditions were used: 95 °C for 15 minutes, 40 cycles at 95 °C for 15 seconds, 60 °C for 1 minute and a dissociation stage (95 °C for 15 seconds, 60 °C for 1 minute, 95 °C for 15 seconds, 60 °C for 15 seconds). Relative levels of mRNA expression were calculated using the Comparative CT/2^−ΔΔCT method [32 (link)] with RNA polymerase II (RPII) as the house-keeping gene for the in-cell-line-based studies [33 (link)].