The geometric mean of GAPDH and PUM1 when used to normalize the expression of CSCs and EMT genes during the validation phase, tumor, nodes, and metastases (TNM) stage III/IV rectal cancer patients showed a 4.79-fold change (P=0.0068) in ALDH1 expression, 1.70-fold change in E cadherin, 4.84-fold change in vimentin (P=0.02), and 11.76-fold change in Twist expression (P=0.003) when compared with stage II rectal tumor when normalized using GAPDH and PUM1 as reference genes. Here, TWIST1 is linked to neoplasm.