Female C57BL/6 mice (6 weeks old, Charles River, Sulzfeld, Germany) were treated with 2% or 0% cholesterol diet (custom product (Altromin, Lage, Germany). The stomach was removed, and the stomach mucosa was scraped off with a sterile scalpel blade on liquid nitrogen until ~2/3 of the tissue was removed.
Female C57BL/6 mice (6 weeks old, Charles River, Sulzfeld, Germany). The stomach was removed, and the stomach mucosa was scraped off with a sterile scalpel blade on liquid nitrogen until ~2/3 of the tissue was removed.
Extracted molecule
total RNA
Label
Cy5
Description
Total RNA was prepared using the RNeasy protocol with on-column DNAse digestion (Qiagen, Hilden, Germany) and confirmed to be intact and free of contaminating DNA using an Agilent 2100 bioanalyzer and a RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA). A custom tailored 'in situ' synthesized 60-mer oligonucleotide microarray (Agilent) comprising 8014 murine genes with immunological focus was used for expression profiling. RNA was amplified and labeled using a reverse-transcriptase/T7 polymerase linear amplification labeling kit (Agilent), and 1.5 mg of respective cRNAs labeled either with Cy3 or Cy5 were hybridized for 16 h at 65°C. Slides were used and processed according to the manufacturer's manuals.
Data processing
Features were extracted with an Image Analysis software (Version A.7.5.1, Agilent). Intensities were normalized by local background correction and by rank consistency filtering with a combination of linear and LOWESS normalization. Data analysis was performed with Rosetta Resolver Biosoftware (Build 5.1.0.0.163, Rosetta Biosoftware, Seattle, WA).
