Quantification of Fibrosis-related Genes

Quantitative Real-time PCR (qRT-PCR) was used to measure mRNA expression levels of αSMA (ACT2A), collagen type I (COL1A1), collagen type III (COL3A1), SMAD2 and SMAD3, as a validation of the array data. All primers were validated (primer efficiency confirmed to be between 90–105%), sequenced and have previously been published^{21 (link),22 (link),61 (link)}. PCR products were run on a 1.5% agarose gel to confirm product size and each product was sequenced to confirm specificity of the primers. Gene expression was quantified using Brilliant SYBR Green QRT-PCR 1-Step master mix (Strategene, the Netherlands) in the Strategene MX3000p system. All expression data were normalized to β-actin using Quantitect primer assay primers (Qiagen, Germany), HS_ACTB_1_SG and corrected using the reference dye ROX. For each experiment >50% of the samples used were also used for the RT² profiler human fibrosis PCR array.