In the model that WNT signaling is suppressed, 3T3-L1 cells were allowed to reach confluence and then exposed to MDI medium for 3 days, which contained 3-isobutyl-1-methylxanthine (0.5 M), dexamethasone (1μM), and insulin (1.75 M), then cultured only in high-glucose DMEM, 10% FBS and insulin alone for the following 2 days, then subsequently cultured with high-glucose DMEM and 10% FBS for 2 days until 7th days of the end of checkpoint. In the model that WNT signaling is activated, Lithium (LiCl) was added into the medium at a concentration of 25mM; To further determine if the WNT signaling was activated, cell was treated with MDI differentiation (i.e. Li+MDI treatment) to see if the cell could differentiate into mature adipocyte.
The 3T3-L1 (ATCC, CL-173TM) pre-adipocyte cell line was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37 ℃ and 5% CO2–air.
Total RNA were extracted using mirVana™ miRNA Isolation (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. The RNA quality was tested with the Agilent Bioanalyzer 2000.
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the Agilent’s miRNA Complete Labeling and Hyb Kit (p/n 5190-0456) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Slides were scanned immediately after washing on the Agilent Scan Control software，using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
microRNA expression in preadipocyte
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.