Primary brown preadipocytes were isolated from young mice 2 weeks according to published methods (Cannon and Nedergaard, 2001; Tseng et al., 2002). When cultured primary brown preadipocytes were at 70-80% confluence, Locked Nucleic Acids (LNAs) miRNA inhibitors (100nM) were transfected by DharmaFect 2 (6ul/ml) according to the manufacturer’s instruction. 24 hrs after transfection, cells were recovered in full culture media and grown to confluence for differentiation for 4 days.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy kits (QIAGEN, Valencia, CA) according to the manufacturer's instructions. DNase (QIAGEN) was added to the RNeasy elution column as recommended by the manufacturer. RNA yield was quantified by UV spectrophotometry, and RNA integrity was verified using an Agilent Bioanalyzer.
Label
biotin
Label protocol
manufacturer's protocol
Hybridization protocol
manufacturer's protocol
Scan protocol
manufacturer's protocol
Data processing
Affymetrix array data were loaded into the R statistical environment with the affy package from BioConductor. Each probe was mapped to an Entrez Gene with R library mouse4302mmentrezgcdf, and genes were normalized with RMA.
