Mice were treated subcutenously for 3 days with 1mg/kg CL 316243 disodium salt dissolved in PBS or PBS as control.
Growth protocol
C57/BL6 mice were kept at room temperature with access to water and standard diet. Cellular fractions containing primary adipocytes and SVF cells were isolated from eight-week-old C57BL/6 female mice by a standard collagenase-based digestion method. Briefly, freshly isolated adipose tissues were minced and digested at 37 °C in digestion medium (0.2% collagenase II, 5% FBS in DMEM) for 30 min with shaking. Mature adipocyte fractions and stromal vascular fractions were separated by incubation on ice for 10 min, followed by centrifugation at 300 g for 10 min. The adipocyte fraction was removed and the SVF was filtered through a 100-µm mesh, followed by washing twice with PBS and filtering through a 40-µm mesh. SVFs were directly used for microRNA isolation.
Extracted molecule
total RNA
Extraction protocol
A QIAzol-Chloroform based protocol was used for the extraction of RNA from tissue samples. Briefly, QIAzol lysis reagent (Qiagen) was added to SVFs. The samples were incubated on ice for 10 min before adding chloroform followed by centrifugation at 7000 g for 20 min. RNA-containing supernatants were mixed with 100% ethanol and subsequently applied to RNA extraction columns provided in the miRNeasy Mini Kit Cat # 217004, Qiagen). To extract RNA, manufacturer’s instructions were followed.
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)in hybridization Oven(Cat#G2545A, Agilent technologies, Santa Clara, CA, US)at 55℃，20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
The chip results were scanned using the Agilent Microarray Scanner (Cat # G2565BA, Agilent technologies, Santa Clara, CA, US) with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) , PMT 100%, 5% were finally normalized using Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US) using Quantile.
Description
microRNA expression after 3d of CL-316,243 treatment
Data processing
Normalization and analysis for differentially expressed genes were performed using robust multi-array analysis and significance analysis of microarrays (SAM) via R statistical software packages “oligo” and “samr”.
