type: obese disease state: with Type 2 Diabetes Mellitus tissue: subcutaneous fat cells
Growth protocol
adipose tissue biopsies were obtained from subcutaneous fat depots during elective surgical procedures (cholecystectomy, surgery of abdominal hernia and gastric by-pass surgery), washed, fragmented and immediately flash-frozen in liquid nitrogen before be stored at -80ºC. These subcutaneous fat samples were provided from a group of 28 women with a body mass index (BMI) between 20 and 55Kg/m2 who were invited to participate at the Endocrinology Service of the Hospital Universitari de Girona Dr. Josep Trueta (Girona, Spain). All subjects were of Caucasian origin and reported that their body weight had been stable for at least three months before the study. They had no systemic disease other than type 2 diabetes mellitus (DM-2) and obesity and all were free of any infections in the previous month before the study. Liver disease and thyroid dysfunction were specifically excluded by biochemical work-up. Other exclusion criteria for those patients included the following: 1) clinically significant hepatic, neurological, or other major systemic disease, including malignancy; 2) history of drug or alcohol abuse, defined as >60g/day, or serum transaminase activity more than twice the upper limit of normal; 3) an elevated serum creatinine concentration; 4) acute major cardiovascular event in the previous 6 months; 5) acute illnesses and current evidence of high grade chronic inflammatory or infective diseases; and 6) mental illness rendering the subjects unable to understand the nature, scope, and possible consequences of the study. All subjects gave written informed consent after the purpose of the study was explained to them. The institutional review board of each institution approved the protocol.
Extracted molecule
total RNA
Extraction protocol
Total RNA, including small RNAs and miRNAs, was extracted, purified and prepared from adipose tissue fragments and cells debris using miRNeasy Mini Kit (QIAgen; Gaithersburg, MD).
Label
Cy3
Label protocol
500ng of total RNA from each sample was dephosphorylated with Calf Intestinal Alkaline Phosphatase (CIP) at 37ºC for 30’ following a denaturalization and then a ligation for 2h at 16ºC using Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies; Palo Alto, CA). In this step a molecule of Cyanine3-pCp is incorporated to the 3’ end of RNA molecules.
Hybridization protocol
Labeled RNA was dried and resuspended with Hybridization Buffer and Blocking Agent, incubated 10’ at 100ºC and transfered to an ice water bath for 5’. Samples were hybridized in a volume of 45ul to the Human miRNA V2 Oligo Microarray (Agilent) for 20 hours at 55°C and 20rpm. Microarrays were then washed at room temperature for 5min in Gene Expression Wash Buffer 1 and 5min at 37ºC in Gene Expression Wash Buffer 2 (Agilent Technologies; Palo Alto, CA).
Scan protocol
Arrays were scanned on an Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies for miRNA microarrays with 100% PMT and 5μm resolution. Data was extracted using Agilent Feature Extraction Software (Agilent Technologies; Palo Alto, CA).
Description
subcutaneous fat cells from obese woman with DM-2, biological replicate 3 of 9
Data processing
Extracted Log2-transformed intensities were quantile normalized to make all data comparable.
