Aims
Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings.
Background & aims
Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings.
Conclusions
RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.
Methods
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.
Results
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. Conclusions: RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.