title: NICHE Human ENL and LNCaP v1.0
status: Public on Nov 01 2007
contact.city: Coleraine
contact.first: Mark
contact.middle: John
contact.last: McCann
contact.email: markjmccann@hotmail.co.uk
contact.state: Northern Ireland
contact.address: Cromore road
contact.department: Northern Ireland Centre for Food and Health
contact.country: United Kingdom
contact.institute: University of Ulster
organism: Homo sapiens
description: The Oligo GEArray® Human Prostate Cancer Biomarkers Microarray profiles the expression of 263 genes useful as molecular markers in the prognosis and diagnosis of prostate cancer.
manufacture_protocol: The LNCaP cell line was obtained from the European Collection of Cell Cultures (ECACC, Salisbury UK) and cultured in RPMI 1640 medium (Gibco Life Technologies, Paisley UK) supplemented with 2.0mM l-glutamine (Gibco), 10.0mM HEPES (Sigma), 1% penicillin-streptomycin (Gibco), 1% of a 45% glucose solution (Sigma), and 10% foetal bovine serum (Gibco), with the media refreshed every ninety-six hours. Enterolactone (CAS 78473-71-9, Fluka, Schelldorf Germany) was prepared at 60μM in cell-line specific media (0.72% DMSO v/v). All control treatments were cell-line specific media adjusted to contain 0.72% DMSO (v/v).
  The cells were grown to 70% confluence as triplicate independent experiments and treated with either enterolactone or media, before incubation for 72 hours under standard cell culture conditions. The total RNA was isolated using RNeasy Mini kit and treated with DNase I (Qiagen, Crawley UK) following the manufacturers guidelines. RNA purity and concentration was measured using the NanoDrop spectrophotometer and all RNA purities were between 1.8 and 2.0.
  The total RNA from treated and untreated cells extracted previously was sent in dry ice by overnight courier to SuperArray Bioscience Corporation (Frederick, USA) for microarray analysis using the Oligo GEArray® Human Prostate Cancer Biomarkers Microarray (OHS-403). The experiment was performed in triplicate. The microarray data was analysed for significance using the students’ t-test, with a value of p=0.05 taken as significant.
  Manufacturer details (ref Superarray)
  Quality Control:
  Suspend in 10 mM Tris pH 8.0 @ > 0.1 mg/ml
  A260/A280 > 2.0 and A260/A230 > 1.7 in Tris pH 8.0
  For Total RNA, 28S:18S agarose gel band intensity ~ 2:1
  Experimental format
  EXP1 Control  and enterolactone
  EXP2 Control  and enterolactone
  EXP3 Control  and enterolactone
  Dye swaping used
  Expression data calculated from mean of experiments.
  Experimental outline available at the SuperArray website (Oligo GEArray Gene Expresssion Array Protocol)
  Agarose Gel or BioAnalyzerElectropherogram