type: obsese day: 7 medium: differentiation tissue: human subcutaneous pre-adipocytes
Treatment protocol
Differentiation was induced using Differentiation Medium (DM; Zen-Bio, Inc.), composed of PM with human Insulin (Ins), Dexamethasone (DXM), Isobutylmethyl-xanthine (IBMX) and PPARγ agonists (Rosiglitazone, Rs). After 7 days (day 7), DM was replaced with fresh Adipocyte Medium (AM; Zen-Bio Inc.), composed of DMEM/Nutrient Mix F-12 medium (1:1, v/v), FBS, HEPES, Biotin, Panthothenate, human Insulin (Ins), Dexamethasone (DXM), Penicillin, Streptomycin and Amphotericin, according to manufacturers’ guidelines.
Growth protocol
Commercially available cryo-preserved human subcutaneous pre-adipocytes from two non-diabetic male subjects with age>40 and Body Mass Index (BMI) <25 or BMI>30Kg/m2 (SP-F-1 or SP-F-3, respectively; Zen-Bio, Inc.) were plated on T-75 cell culture flasks and cultured at 37ºC and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mix F-12 medium (1:1, v/v) supplemented with Fetal Bovine Serum (FBS) 10%, HEPES 1%, Glutamine 1% and Penicillin/Streptomycin (P/S) at 10U/mL (all from GIBCO, BRL; Grand Island, NY). One week later, human subcutaneous pre-adipocytes were resuspended and cultured (~40.000cells/cm2, 3rd passage) in 6-well plates with Pre-adipocyte Medium (PM; Zen-Bio, Inc.) composed of DMEM/Nutrient Mix F-12 medium (1:1, v/v), FBS 10%, HEPES 1%, Glutamine 1% and P/S 1% in a humidified 37ºC incubator with 5% CO2. Twenty-four hours after plating, cells were checked for complete confluence (day 0). Two weeks after the initiation of differentiation (day 14), cells appeared rounded with large lipid droplets apparent in the cytoplasm. Cells were then considered mature adipocytes (MAs).
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
pre-adipocytes of obese subject after 7 days of differentiation, replicate 3 of 3
Data processing
Extracted Log2-transformed intensities were quantile normalized to make all data comparable.
