title: MWG BIOTECH 11K ARRAY
status: Public on Jun 01 2006
contact.city: Boston
contact.first: Winston
contact.middle: Patrick 
contact.last: Kuo
contact.email: wkuo@genetics.med.harvard.edu
contact.state: MA
contact.address: 188 Longwood Avenue
contact.department: Genetics
contact.country: USA
contact.institute: Harvard Medical School
organism: Mus musculus
manufacture_protocol: Briefly, RNA was prepared using the RNeasy Midi kit according to the manufacturer's instructions (Qiagen). First-strand complementary DNA synthesis from 5ug total RNA was done at 42oC for 1h, followed by second-strand cDNA synthesis at 16oC for 2h. RNase I digestion was followed by proteinase K treatment. The resulting double-stranded cDNA was purified using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). Fluorescence-labeled RNA was generated by carrying out an in vitro transcription reaction (40 mM Tris-HCl, pH 7.5, 7 mM MgCl2, 10 mM NaCl, 2 mM spermidine, 5 mM ditihothreitol, 7.5 mM each of ATP, CTP and GTP, 5 mM UTP, 20 units RNase inhibitor and 1,000 units T7 polymerase) at 37oC for 4h. This reaction was done in the presence of fluorescence-labeled nucleotide (Amersham Pharmacia Biotech) to generate Cy3- or Cy5-labelled RNA. The labeled RNA was subsequently purified (Qiagen RNeasy Mini kit) and chemically fragmented at 94oC for 15 min in fragmentation buffer (20 mM Tris-acetate, pH8.1, 50 mM potassium acetate, 15 mM magnesium acetate). The fragmented, Cy3- or Cy5-labelled complementary RNA (20ug) was lyophilized, resolubilized in hybridization buffer (50% formamide, 50mM sodium phosphate, pH8.0, 6X SSC, 5X Denhardt's Solution, 0.5% SDS), and hybridized to oligonucleotide mouse 11K microarrays at 42oC for 24h. T7 RNA polymerase was purchased from Ambion: primers, RNase inhibitors and all other enzymes were purchased from Roche Molecular Biochemicals.