Brown adipose tissue, inguinal adipose tissue, epidydimal adipose tissue, gastrocnemius muscle, and soleus muscle were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. RNasey Mini Kit(Qiagen p/n 74104) was used to obtain 1 ug of total RNA for miRNA labeling.
Label
Cy3
Label protocol
miRCURYTM Array Power Labeling kit (Cat #208032-A, Exiqon) was used for miRNA labeling.
Hybridization protocol
miRNA array hybridization was performed using miRCURYTM Array Hybridization buffer and Wash buffer
Scan protocol
Axon GenePix 4000B microarray scanner and GenePix pro V6.0 were used for miRNA array scanning and analysis.
Data processing
GenePix pro V6.0 is used to read the raw intensity of the image. The intensity of green signal is calculated after background subtraction and four Replicated spots of each probe on the same slide have been calculated the median. We use Median Normalization Method to obtain “Normalized Data”, Normalized Data = (Foreground-Background) / median, the median is 50 percent quantile of microRNA intensity which is larger than 50 in all samples after background correction.
