Quantitative RT-PCR Analysis of Gene Expression

Total RNA from tissue samples and from cell-line pellets was extracted using TRIzol Plus RNA Purification System (Life Technologies, Paisley, UK), and quantified by NanoDrop ND-1000 (Thermo Fisher Scientific, Loughborough, UK). Total RNA was reverse transcribed using AMV First Strand cDNA synthesis kit (New England Bio Labs, Hertfordshire, UK) after DNase treatment (DNase I (#M0303), New England Bio Labs, Hertfordshire, UK), using the manufacturer’s protocol as previously described [34 (link)]. cDNA was amplified by qPCR using JumpStart SYBR Green supermix (Sigma, Dorset, UK) and CFX Connect Real-Time System (Bio-Rad, Hertfordshire, UK) and the following primers: AGR2 amplification were: forward 5′-ATTGGCAGAGCAGTTTGTCC-3′, reverse 5′-GAGCTGTATCTGCAGGTTCGT-3′ [45 (link)]; for Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ), forward 5′-CGTTACTTGGCTGAGGTTGCC-3′, reverse 5′-GTATGCTTGTTGTGACTGATCGAC-3′ [46 (link)]; and for Peptidylprolyl Isomerase A (PPIA), forward 5′-AGACAAGGTCCCAAAGAC-3′ and reverse 5′-ACCACCCTGACACATAAA-3′ [47 (link)]. Relative transcript expression was calculated using the ΔΔCT method, normalised to the reference genes YWHAZ and PPIA using Biorad CFX manager.