title: CHUGAI 41K
status: Public on Jan 26 2004
contact.city: Koto-ku
contact.first: Yuichi
contact.last: Ishikawa
contact.email: ishikawa@jfcr.or.jp
contact.state: Tokyo
contact.address: 3-8-31 Ariake
contact.country: Japan
contact.institute: Cancer Insitute
contact.phone: 81 3 3570 0488
organism: Homo sapiens
description: Patients and Sample Preparation.
  We analyzed NT samples from 38 patients undergoing surgery at the Cancer Institute Hospital, Tokyo, during May 1996 – May 2001. All samples were collected with informed consent and ethical committee approval and were grossly dissected, snap-frozen in liquid nitrogen within 20 minutes of removal. Initial diagnosis of each sample  from frozen section was later confirmed by detailed analysis of paraffin sections and immunohistochemistry. Following WHO guidelines,1 17 tumours were classified as SCLC including two combined SCLC with adenocarcinoma (SCLC-AD), 8 as LCNEC, including 2 combined LCNEC with AD (LCNEC-AD), 12 as TC and 1 as AC. The prevalence of LCNEC as a percentage all lung cancer resected at the Cancer Institute during the period was 2.7%. Since LCNEC is described as a variant of large cell carcinoma MAGE(LCC), we also analyzed thirteen LCC samples. Twelve well differentiated  AD samples were included to aid interpretation of expression profiles for the SCLC-AD and LCNEC-AD samples, and to allow identification of markers that distinguish NT from non-small cell lung cancer. We included thirty normal lung samples from unrelated individuals. Finally 11 SCLC cell lines were incorporated into the analysis. Full clinical details of all tumour samples and cell lines are provided in supplementary table 1. Reference RNA, a mixture of lung cancer cell lines and normal lung, is exactly as described previously.5 Cell lines were obtained from the American Type Culture Collection. the Japanese Collection of Research Bioresources, or The RIKEN cell bank.
  cDNA microarray
  Each microarray contains 40,368 elements, of which 39,936 are derived from IMAGE cDNA clones purchased from Research Genetics (Huntsville, Alabama). This clone set was supplemented with 384 proprietary clones and 48 control elements. According to our latest estimate, the array represents 29,487 unique UNIGENE clusters accounting for 9,085 named genes, 15,276 ESTs and 5126 other un-named genes (for example, clones described as hypothetical proteins). Clones were purchased as sequence verified, but we estimate an error rate of at least 10%. Genes mentioned by name in this paper have been re-sequenced. cDNA PCR products were resuspended in 50% DMSO and printed at a pitch of 150 µm onto CMT-GAPS slides (Corning, Corning, NY) using a MicroGrid II spotting robot fitted with 10K pins (BioRobotics, Cambridge, UK). Relative humidity was controlled within the range 40-50% and temperature between 20-25°C.
  
  cDNA derived from 2µg of cRNA, essentially as described,7 was aminoallyl labeled8 with Cy5 (sample) or Cy3 (reference), hybridized overnight at 42ºC in a buffer containing 50% formamide, 5xSSC, 0.1% SDS, 0.25µg/µl human cot1 DNA and 0.125µg/µl poly dT and washed to a final stringency of 1x SSC at 42°C. After washing, slides were immediately scanned using an Axon GenePix 4000B scanner and quantified using Axon GenePix Pro software (Axon, Union city, CA).  Raw data for all 40,386 elements across all 105 samples assayed here is deposited with