group: after weight loss gender: Male age: 33 bmi: 38.3
Treatment protocol
Subcutaneous abdominal adipose tissue biopsies were obtained by needle aspiration under local anesthesia (2 ml 10% lidocain). Biopsies were immediately put in RNAlater (Ambion, Austin, TX) and after overnight incubation at 4C snap frozen at -80C until further processing.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Tri reagent (Sigma-Aldrich, St. Louis, MO) followed by RNeasy Midi kit (Qiagen, Düsseldorf, Germany). The RNA was further concentrated by RNeasy MiniElute (Qiagen, Düsseldorf, Germany) and SpeedVac (DNA 120 SpeedVac, Thermo Savant, Waltham, MA 02454).
Label
biotin
Label protocol
Performed according to manufacturer’s recommendation at the SCIBLU Microarray Resource Centre, Lund University, Sweden
Hybridization protocol
Performed according to manufacturer’s recommendation at the SCIBLU Microarray Resource Centre, Lund University, Sweden
Scan protocol
Performed according to manufacturer’s recommendation at the SCIBLU Microarray Resource Centre, Lund University, Sweden
Data processing
We used AffyProbeMiner to regroup the individual probes into consistent probe sets and remap the probe sets to the correct sets of mRNA transcripts for HG-U133 Plus 2.0 array which resulted in 23,798 probe sets. We applied Robust Multi-array Average (RMA) method for normalization. We conducted filtering based on the MAS5.0 present/absent calls which classified each probe set as expressed above background (present call) or not (absent or marginal call). We included probe sets, which have detection call as present call in at least 50% arrays in one group, which left 13,463 probe sets out of 23,798 for further analysis. To identify differentially expressed genes for interclass paired comparisons we used a permutation based false discovery rate (FDR) approach calculated by the EOC function in the OCplus R package as well as Patterns from Gene Expression (PaGE) method for log (base 2) transformed data with the use of modified t statistics. We considered only those probe sets as differentially expressed between groups that fulfilled each of two requisites: (i) a global FDR less than 0.05 by the EOC function, and (ii) a confidence level of at least 0.95 (which is equivalent to 5% of false discovery rate by PaGE method. Gene lists were analyzed using WebGestalt for the enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the hypergeometric test.
