tissue: Brown adipocytes differentiation status: pre strain: NMRI
Treatment protocol
The cells were cultured in 10 cm2 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum (NCS) (Hyclone, Erembodegem, Belgium, or Invitrogen, Carlsbad, CA), 0.17 mM insulin, 25 g/ml sodium ascorbate, 10 mM Hepes (pH 7.4), 4 mM glutamine, 50 units/ml penicillin and 50 g/ml streptomycin, supplemented or not with 1 mM rosiglitazone maleate (Alexis Biochemicals, Lausen, Switzerland). The media were changed on day-1, day-3, and day-5 of culture. Some of the cell cultures, grown in absence of rosiglitazone, were treated with 1 mM norepinephrine (Sigma–Aldrich, Stockholm, Sweden) 2 h prior to harvest
Growth protocol
Brown adipocytes we harvested from male NMRI mice housed under standard conditions. At the age of 4 weeks, the mice were killed and cells from pooled interscapular, cervical and axillary brown adipose tissue depots. Epididymal white adipose tissue were isolated from the same mice.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cultured cells with Ultraspec (Biotecx, Houston, TX) according to the manufacturer’s protocol
Label
Hy3
Label protocol
Samples were labelled using Exiqon’s miRCURY LNA array labelling kit (Vedbæk, Denmark): 2ug total RNA was incubated with labelling buffer, the Hy3 dye, labelling enzyme and spike-in microRNAs for 1 hour at 37°C. The enzyme was then heat-inactivated at 65°C for 15 minutes, followed by addition of 2 x hybridisation buffer. The samples were incubated at 95°C for 5 minutes while protected from light. The samples were then briefly spun down and filtered through a 0.45-micron durapore filter (Millipore), and 40 ul sample was loaded to the arrays by capillary force using a cover slip (Erie Scientific).
Hybridization protocol
The arrays were incubated at 60°C for 16 hours in a hybridisation oven.
Scan protocol
After hybridisation the slides were manually washed for 2 minutes in Buffer A at 60oC, rinsed in Buffer B followed by a 2-minute wash in buffer B at RT and 2 minutes in Buffer C at RT. All buffers were supplied by EXIQON. After washing the slides were dried by centrifugation for 2 minutes at 1000rpm. The slides were immediately scanned using an Agilent G2565BA scanner with a spot size of 10um and 100% PMT gain.
Data processing
QuantArray software was used to quantify the signals from the Agilent scanner using the fixed circle approach. Background subtracted signal intensities were normalized in R using quantile normalization.
