Sybr green probe

Total RNA was isolated from tissue and cells using a Directzol kit (Genesee Scientific; El Cajon, CA). The gene expression was measured using TaqMan probes (Applied Bio-systems, Foster City, CA) or SYBR Green probes (Thermo Fisher Scientific) on LightCycler96 (Roche Diagnostics, Indianapolis, IN). Liver NHE8, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and Lgr5 were measured using the TaqMan reaction system. Collagen 1α (Col 1α) was measured using the SYBR Green system. TATA-binding protein (for TaqMan probes) and β-actin (for SYBR Green probes) were used as an endogenous reference to normalize gene expression levels.

Quantitative RT-PCR was performed to validate gene expression changes identified by RNA sequencing. Briefly, total RNA was extracted from drug-treated HCC827-ER3 cells using the RNeasy mini kit (Qiagen, Valencia, CA). cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT) primers (Life Technologies, Carlsbad, CA), and RT-PCR was performed on a LightCycler with SYBR Green probes (Thermo Scientific) according to the manufacturer’s protocol. Ratios of the expression level of each gene to that of the reference gene were then calculated. Sequences for the primers used for quantitative RT-PCR for the selected genes are listed in Table S1.

After 1, 6, 12, and 24 hours of cell co-culture, the medium was removed and cells were washed with phosphate-buffered saline. Total RNA was isolated from cells and tissue biopsy specimens using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Reverse transcription was performed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time quantitative polymerase chain reaction (qPCR) for mRNA expression was performed using SYBR Green probes and an ABI 7500 thermal cycler (Applied Biosystems). The following primers were used, based on a previous study. TRPV1 forward, 5′-GAGTTTCAGGCAGACACTGGAA-3′; TRPV1 reverse, 5′-CTATCTCGAGCACTTGCCTCTCT-3′; GDNF forward, 5′-CTTGGGTCTGGGCTATGAAAC-3′; GDNF reverse, 5′-CAAAGGCGATGGGTCTGC-3′; NGF forward, 5′-AGCAAGCGGTCATCATCC-3′; and NGF reverse, 5′-GTGGCGGTGGTCTTATCC-3′. GAPDH was used as an endogenous reference. The amplification protocol consisted of an initial denaturation step at 95 °C for 10 seconds, followed by 40 cycles of denaturation for 5 seconds at 95 °C and annealing/extension for 33 seconds at 60 °C. Relative expression levels were normalized by dividing the target gene Ct values by the endogenous GAPDH Ct values.