Agilent mirna microarray 15 k

For mammary gland miRNAs, the synthesis of target miRNA probes and hybridization were performed using Agilent's miRNA Labelling Reagent and Hybridization kit (Agilent Technology, USA) according to the manufacturer's instructions. Briefly, 100 ng of total RNA from each region (the 3^rd mammary gland and the inter-mammary gland region) at E13.5 was dephosphorylated with ∼15 Units of calf intestine alkaline phosphatase (CIP), followed by RNA denaturation with ∼40% DMSO and a 10 min incubation at 100 °C. Dephosphorylated RNA was ligated with pCp-Cy3 mononucleotide and purified with MicroBioSpin 6 columns (Bio-Rad, USA). After purification, labelled samples were resuspended with Gene Expression blocking Reagent and Hi-RPM Hybridization buffer, followed by boiling for 5 min at 100 °C and 5 min chilled on ice. Finally, denatured labelled probes were pipetted onto the assembled Agilent miRNA Microarray (15K) and hybridized for 20 hours at 55 °C with rotation at 20 rpm in an Agilent Hybridization oven (Agilent Technology, USA). The hybridized microarrays were washed as the manufacturer's washing protocol (Agilent Technology, USA). Location of array raw data; GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66546)