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Molecular subsets in the gene expression signatures of scleroderma skin

A. Milano et al.

  • Patients
    • Patient Details
  • Data
    • cDNA synthesis, microarray hybridization and data processing
  • Intrinsic
    • Selection of intrinsic gene and hierarchical clustering
  • Statistics
    • Robustness and statistical significance of clustering
  • PCA
    • Principal component analysis
  • Module Maps
    • Module maps using Genomica
  • Clinical Params
    • Correlation to clinical parameters
  • IHC
    • Immunohistochemistry

cDNA synthesis, microarray hybridization and data processing

200 ng of total RNA from each biopsy was converted to Cy3-CTP (Perkin Elmer) labeled cRNA, and Universal Human Reference (UHR) RNA (Stratagene) was converted to Cy5-CTP (Perkin Elmer) using a low input linear amplification kit (Agilent Technologies). Labeled cRNA targets were then purified using RNeasy columns (Qiagen). Cy3-labeled cRNA from each skin biopsy was competitively hybridized, along with Cy5-CTP (Perkin Elmer) labeled cRNA from Universal Human Reference (UHR) RNA pool (Stratagene), to 44,000 element DNA oligonucleotide microarrays (Agilent Technologies) representing more than 33,000 known and novel human genes in a common reference design [16]. Hybridizations were performed for 17 hours at 65oC with rotation.

After hybridization, arrays were washed following Agilent 60-mer oligo microarray processing protocols (6 X SSC, 0.005% Triton X-102 for 10 min. at room temperature; 0.1 X SSC, 0, 005% Triton X-102 for 5 min at 4oC, rinse in 0.1 X SSC). Microarray hybridizations were performed for each RNA sample resulting in 61 hybridizations. Fourteen replicate hybridizations were added, resulting in a total of 75 microarray hybridizations.

Microarrays were scanned using a dual laser GenePix 4000B scanner (Axon Instruments). The pixel intensities of the acquired images were then quantified using GenePix Pro 5.0 software. Arrays were visually inspected for defects or technical artifacts, and poor quality spots were manually flagged and excluded from further analysis. Only spots with fluorescent signal at least twofold greater than local background in both Cy3- and Cy5- channels were included in the analysis. Probes missing more than 20% of their data points were excluded, resulting in 28,495 probes that passed the filtering criteria. The data were displayed as log2 of the LOWESS-normalized Cy5/Cy3 ratio. Since a common reference experimental design was used, each probe was centered on its median value across all arrays.

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