Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from 10 patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or X-ray contrast solution on RNA quality.
RNA was of variable quantity (100-1500 ng) and poor quality (Agilent RNA Integrity Number (RIN) <5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130 bp. Differential gene expression by microarray analysis identified 1140 up-regulated genes and 1001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 up-regulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC, and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r = 0.41 to r = 0.57). Immunohistochemistry of surgical resection tissues (n = 23) showed elevated CD9, SERPINA3, and PNMA2 protein expression in cancer samples.
RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.