Quantitative Gene Expression Analysis of CRC Samples

RNA extraction from the cell lines, xenograft animal tumors, and CRC-derived organoids, treated with DMSO (vehicle), OPCs, andrographis and their combination, was performed using the MiRNeasy Mini Kit (Qiagen); followed by conversion to cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA) as previously described^{17 (link)}. cDNA derived from 5 ng of RNA was used and qRT-PCR was performed using SensiFAST SYBR mix (Bioline, London, UK) using the primer sequences listed in Supplementary Table S1. Briefly, cDNA samples were mixed with 0.5 µl of 10 µM each of forward and reverse primers specific for the target genes, 5 µl SYBR green master mix and volume was made up with nuclease-free water. The relative expression for target genes was calculated using 2^−ΔΔCT method normalized against the housekeeping β-actin gene.