Microrna complete labeling and hyb kit

Total RNAs were extracted from cells lysed in Trizol (Invitrogen) according to the manufacturer’s instruction. For the microRNA microarray, microRNA Complete Labeling and Hyb kit (Agilent Technology, GE5190-0456) was used, following the manufacturer’s instruction. Briefly, 100 ng of total RNA were incubated with CIP at 37°C for 30 min. Dephosphorylated RNA sample was denatured with DMSO at 100°C for 5 min. Ligation master mix was prepared following the manufacturer’s instructions (10×T4 RNA ligase buffer, Cyanine3-pCp, T4 RNA ligase) and added to each sample. Samples were resuspended with blocking agent and Hi-RPM hybridization buffer, and incubated at 100°C for 5 min, and then loaded onto Agilent’s Human microRNA microarray (Agilent Technology, G4112F). Human miRNA Microarray Kit (Aglent Technology, G2545A) was used for hybridization. Samples were hybridized at 55°C and 20×rpm for 20 h on Agilent Hybridization oven (Agilent Technology, G2545A). The hybridized microarrays were washed following the manufacturer’s washing instructions (Agilent Technology).