Patients had been fasting over night. An abdominal subcutaneous adipose tissue needle biopsy was obtained under local anesthesia. The biopsy was rapidly rinsed in saline and snap frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the Nucleospin RNA11 kit (Macherey-Nagel, Duren, Germany). RNA samples were treated with RNase-free DNase (Macherey-Nagel).
Label
biotin
Label protocol
Total RNA were amplified and biotin labeled using the Whole Transcript (WT) Sense Target Labeling Protocol.
Hybridization protocol
From non-degraded high-quality total RNA we prepared and hybridized biotinylated complementary RNA to Gene 1.1 ST Arrays, and then washed, stained and scanned slides using standardized protocols (Affymetrix, Inc., Santa Clara, CA).
Scan protocol
We used standardized Affymetrix protocols (Affymetrix, Inc., Santa Clara, CA).
Data processing
Subsequent data analyses were performed using the Affymetrix GeneChip Operating Software (GCOS) Version 1.4. To allow comparisons of transcript levels between samples, all samples were subjected to an all-probe set scaling to target signal 100. # Summarization: PLIER, Background correction: PM-GCBG, Normalization method: Global Median.
