title: Zhejiang university human 449 oligonucleotide array
status: Public on Nov 24 2009
contact.city: Hangzhou
contact.first: youying
contact.last: mao
contact.address: Yuhangtang Road,NO.388
contact.country: China
contact.institute: Zhejiang University
organism: Homo sapiens
description: Preparation of microarray: 449 immune related genes were included. Specific 70mer oligonucleotides of each gene was searched in the human_V4.0.1_genelist_s+ (Qiagen-Operon).Another 10 housekeeping genes were also included. Oligos (1OD) were dissolved in 100 ul ddH2O and printed on Immobilon-Ny+ Transfer T Membranes (Millipore, Bedford, MA, USA) using GeneTAC M G3 arrayer (Genomic Solutions, Ann Arbor, MI, USA). Each oligo was duplicated triple in each array to verify the reproducibility of experiments.
  Preparation of labeled cDNA probe: The RNA samples were labeled with 33P-dCTP during the first-strand reverse transcription reactions. The labeling reaction contained 20mmol/L each of dATP, dTTP and dGTP, 10mmol/L dCTP and 3ul of alpha-33P-dCTP (10mCi/ml, Amersham Biosciences, Buckinghamshire, UK), 10ul of First-Strand Buffer, 0.5mmol/L DTT and 400 unit SuperScriptTM II RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 0.3 ug oligo(dT)16 primer, 0.1 ug random hexamer primer and total RNA at the optimal concentration in a reaction volume of 50 ul. The reaction proceeded at 42C for 2 hours and products were purified using a G-50 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
manufacture_protocol: see manufacturer's web site