title: Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Chapman
authors.forename: Robert
authors.initials: R
authors.lastname: Tidswell
authors.forename: James S
authors.initials: JS
authors.lastname: Dooley
authors.forename: Neomal S
authors.initials: NS
authors.lastname: Sandanayake
authors.forename: Virginie
authors.initials: V
authors.lastname: Cerec
authors.forename: Maesha
authors.initials: M
authors.lastname: Deheragoda
authors.forename: Alvin J X
authors.initials: AJ
authors.lastname: Lee
authors.forename: Charles
authors.initials: C
authors.lastname: Swanton
authors.forename: Fausto
authors.initials: F
authors.lastname: Andreola
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Pereira
mesh_terms: D000328:Adult; D000368:Aged; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D001650:Bile Duct Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D001653:Bile Ducts, Intrahepatic; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D018281:Cholangiocarcinoma / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D002760:Cholangiopancreatography, Endoscopic Retrograde / Q000379:methods*; D005260:Female; D020869:Gene Expression Profiling; D055106:Genome-Wide Association Study; D006801:Humans; D008297:Male; D046228:Microarray Analysis; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies; D015843:Serpins / Q000235:genetics / Q000378:metabolism; D060245:Tetraspanin 29 / Q000235:genetics / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C119249:Ma2 antigen; D009419:Nerve Tissue Proteins; D012334:RNA, Neoplasm; C516148:SERPINA3 protein, human; D015843:Serpins; D060245:Tetraspanin 29