title: MIT-Laboratory for Bioinformatics and Metabolic Engineering, 17K Mouse Arrays
status: Public on Apr 14 2003
contact.city: Cambridge
contact.first: R.
contact.middle: Michael
contact.last: Raab
contact.email: rmraab@mit.edu
contact.state: MA
contact.address: 77 Massachusetts Avenue
contact.department: Chemical Engineering
contact.country: USA
contact.web_link: http://web.mit.edu/cheme/gnswebpage/index.shtml
contact.institute: Massachusetts Institute of Technology
contact.phone: 617-258-0349
organism: Mus musculus
description: Total RNA samples are generally isolated using either STAT60
  or Qiagen's QIAquick Total RNA mini-kit.
  
  Total RNA control samples are labelled using Cy3 dCTP (Perkin-Elmer), and
  total RNA experimental samples were labelled using Cy5 dCTP (Perkin-
  Elmer) during reverse transcription. For reverse transcription 10 ug of total
  RNA was mixed with 2 uL of 10X Cy-labelled dCTP, 2 uL of 100 mM DTT
  (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP
  mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen),
  2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II
  reverse transcriptase (Invitrogen). The control RNA samples were additionally
  mixed with 2 uL of random hexamers. The reaction mixtures were incubated
  at 42 C for two hours. After the reverse transcription reactions, the RNA
  templates were degraded by addition of 2 uL of 1 N NaOH and incubation at
  65 C for 10 minutes. The alkaline conditions were neutralized by addition of
  2 uL of 1 N HCl. Experimental cDNA samples were then mixed with a control
  cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal
  kit (Qiagen) as described by the manufacturer's instructions. The cleaned
  samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at
  65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech
  Laboratories), quantified, and hybridized to prepared oligonucleotide
  microarrays.
  
  Hybridization occurred overnight at 55 C using Corning
  hybridization chambers. The following day the arrays were washed in Glass
  Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and
  finally in 0.1X SSC. The arrays were dried by centrifugation and scanned
  using a GenePix 4000B scanner (Axon Instruments). The resulting data is
  downloaded and formatted in Excel (Microsoft), and analyzed using Matlab
  (The MathWorks, Inc.).
  
  These microarrays were prepared using GAPS glass slides (Corning), and a
  Virtek arrayer (Bio-Rad). Arrays contained 17,280 features, printed from
  a synthesized oligonucleotide mouse library (Operon). The library was
  suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were
  printed at approximately 50% humidity. Once printed, the probes were
  crosslinked using a UV Stratalinker (Stratagene). The slides were then
  blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic
  anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and
  10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by
  rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol
  (Pharmco Products). After blocking, they were dried using filtered air
  and stored in the dark at room temperature.
  
  ARRAY VALIDATION. Microarray printing and hybridization protocols
  were validated in our laboratory. For validation, we prepared arrays
  containing an approximately 13,000 gene sub-set of our oligonucleotide
  mouse library, printed in triplicate. Total RNA from fetal skeletal muscle
  and fetal brain were used for validation. Each sample contained an equal
  amount of RNA from the two sources. Validation experiments compared muscle
  RNA versus muscle RNA and brain RNA versus muscle RNA. Each sample was prepared as
  described above and conducted in duplicate.
  The arrays were processed, scanned, and the data obtained from the
  image files. Matlab was used to calculate basic statistics.
  The analysis required as input the gene identifiers from the Genepix
  (scanner) data output, the ratios of means of the Cy5/Cy3 pixel fluorescence
  corresponding to each gene identifier, the ratio of means normalization factor
  (used to correct for the difference in fluorescence and incorporation of the
  Cy5 and Cy3 fluorophores), and the adjusted flags corresponding to each
  gene identifier. The adjusted flags marked features that either did not possess
  basic fluorescence requirements sought by the imaging software or had
  less than 60% of their pixels' intensities greater than two standard deviations
  of the background intensity over the background intensity. Using these data
  as input, Matlab returned the gene identifier, the mean of replicate spots that
  were not flagged by our filters, the standard deviation of the unflagged
  replicates, the coefficient of variation among replicates, and the number of
  observations used in calculating the statistics for each gene.
  The coefficient of variation, CV, was calculated for each replicated gene expression.
  For the muscle versus muscle control arrays, the median CV across all
  probes was 10.2%. For the muscle versus brain arrays the median coefficient
  of variation across all probes was 9.8%. This indicates that for a gene
  transcription ratio of 1, we might expect the true value to lie between 0.9 and
  1.1; similarly for a gene transcription ratio of 3, we might expect the true
  value to lie between 2.7 and 3.3.
  In duplicate arrays, 76% of the genes observed on one muscle versus muscle
  array were also observed on the duplicate; likewise 77% of the genes found
  on one muscle versus brain array were conserved on the duplicate. These data
  demonstrate the inter-array reproducibility by showing the majority of genes
  are reproducibly found in multiple replicate arrays.
  Keywords = mouse, dna microarrays, diabetes, hepatoma, hepatocytes, liver, muscle, adipose, hypothalamus