Nanodrop

Total RNA was isolated from 3D-HT-29 DMSO control, dabrafenib-resistant 3D-HT-29 Replicates A, B and C; 3D-HCT-116 DMSO control, irinotecan-resistant 3D-HCT-116 Replicates A, B and C cells using the Invitrogen PureLink RNA Mini Kit (Thermo Fisher Scientific, USA) according to manufacturer’s instructions. The concentration and purity of the isolated RNA samples were determined using the NanoDrop instrument (Biodrop, UK). Complementary DNA (cDNA) synthesis was performed by using 1 μg total RNA, 5 μM oligo d(T), 1X ProtoScript II Reaction Mix, and 1X ProtoScript II Enzyme Mix as a final concentration (ProtoScript II First Strand cDNA Synthesis Kit, New England BioLabs, USA).
Real time RT-PCR reaction was performed using GoTaq® qPCR Master Mix (Promega, USA). GAPDH gene was used as a housekeeping gene for the analysis. Each reaction was carried out in three biological replicates. Data were displayed as fold changes and analysed using the 2^−ΔΔCt method [48 (link)]. The list of primers used are as following (Table 1).

We pooled two samples to obtain the PBMCs. The genomic DNA was extracted from four sets of cultured PBMCs using a DNA extraction kit (BiotechRabbit, Germany) following the manufacturer’s instructions. DNA concentration was determined using spectrophotometry at a wavelength of 260 nm and DNA purity was determined using OD₂₆₀/OD₂₈₀ using the NanoDrop (Biodrop, UK). Polymerase chain reaction was conducted using a standard protocol (Green PCR MasterMix, BiotechRabbit) but with a different annealing temperature for each primer (TLR4 and β-actin) (Table-1) [13 (link)]. The β-actin gene was used to assess the quality of genomic DNA samples. The PCR reaction mix (12.5 μL) contained 1 μL genomic DNA, 1.25 μL primers, 5 μL ddH₂O, and 6.25 μL GreenPCR Master Mix(Qiagen, Hilden, Germany). Initial denaturation was performed at 95°C for 5 min, followed by 35 cycles at 95°C for 1 min. The annealing temperature was at 59.5°C for TLR4 and 60.5°C for β-actin for 25 s with a 10 min final extension step at 72°C. The negative control consisted of each sample without the genomic DNA. The PCR products were stored at 4°C, and 10 μL was used for electrophoresis (1.5% agarose gel). The DNA bands were observed under UV irradiation (Gel Doc XR system, Bio-Rad, California,USA).