Sensifast sybr mix

cDNA was synthesised using Bioscript™ Reverse Transcriptase and random hexamers (Bioline Pty Ltd, Australia) according to the manufacturer’s protocol. This was the same RNA used for the microarrays. Quantitative PCR (qPCR) was used to validate genes found to be differentially expressed on microarray. Beta Actin (bAct), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), and hypoxanthine guanine phosphoribosyl transferase (Hprt1) were used as normalising genes (Additional file 1: Table S1). Individual reactions (10 μl) contained 2× Sensifast SYBR Mix (Bioline), forward and reverse primers (10 μM), and 4 μl of cDNA (or 4 μl water for no template controls). The PCR reactions were carried out in a MxPro3005P Real Time PCR System (Stratagene, Agilent Technologies, USA) followed by a dissociation curve. All samples were run in triplicate.
Cycle threshold (Ct) values for each sample were calculated using the MxPRo QPCR software (Stratagene, Agilent Technologies, USA). Triplicate Ct values were averaged and the quantity (Q) of each sample was calculated using the delta-delta Ct method. Q values from the normaliser genes were input into geNorm and the geometric means from these genes were used to generate a normalisation factor (NF) [9 ]. The Q values of the genes of interest were normalised by dividing by the NF value.

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.

RNA from HCT116, SW480, SW620, RKO, LoVo and HT29 cells treated for 18 hours or tumor organoids treated for 7 days with DMSO (vehicle), OPCs (100 ng/ul), curcumin (1 ng/ul), OPCs(100 ng/ul) + curcumin(1 ng/ul) and OPCs(25 ng/ul) + curcumin(0.5 ng/ul), were isolated using mRNeasy kit (Qiagen). RNA from mice xenograft tumors collected in RNAlater solution (Qiagen) were extracted using mRNeasy kit (Qiagen) following the manufacturer’s instructions. Extracted RNA was used as a template for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to manufacturer’s protocol. RT-qPCR was performed using SensiFAST SYBR mix (Bioline, London, UK) using the primer sequences listed in Supplemental Table 2. All RT-qPCR target genes were calculated using ΔΔCt method normalized to β-actin.