Genechip mirna array 3

Affymetrix^® GeneChip^® miRNA Array 3.0 was conducted to detect the expression of miRNAs in MCF7-Pa and MCF7-Re cells. After cultured in RPMI 1640 medium without phenol red supplemented with 5% cFBS and without tamoxifen, total RNA was harvested using TRIzol^® reagent (Life Technologies, USA) according to the manufacturer's instructions. RNA extraction and miRNA profile detection was provided by CapitalBio Corporation (Beijing, China). Raw data were analyzed using expression console software version 1.2 using default analysis settings and RMA as normalization method. MiRNAs with values above the cutoff of 10 in all samples were chosen for data analysis. Differentially expressed miRNAs were identified through Fold Change filtering. Microarray data were deposited to GEO database with the accession code GSE66607.

Total RNA including low molecular weight RNA was labeled using the Flashtag RNA labeling kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, for each sample, 400 ng of total RNA was subjected to a tailing reaction followed by the ligation of the biotinylated signal molecule to the target RNA sample. Each sample was hybridized to a GeneChip® miRNA Array 3.0 (Affymetrix, Santa Clara, CA, USA) for 16 h at 48 °C at 60 rpm. After washing and staining with a Fluidics Station 450 (Fluidics script FS450_0002), arrays were scanned with a GeneChip® Scanner 3000 7G (Affymetrix). The image data were analyzed with the Expression Console Software (www.affymetrix.com) for quality control. GeneChip® miRNA Array 3.0 contained 19,724 total mature miRNA probe sets and covered 153 organisms. This array contained 1111 and 855 mouse mature miRNA and pre-miRNA, respectively. miRNA probes of this array were derived from the Sanger miRBase miRNA database v17.