Mircury array wash buffer kit

Expression levels of miRNAs in moDCs, LPS-conditioned moDCs and TGF-β-conditioned moDCs were analyzed using Exiqon microRNA arrays (Denmark). Total RNA was isolated using the TRIZOL® Reagent (Invitrogen Life Technologies). The concentration and purity of the RNAs were determined using the NanoDrop® ND-1000. The miRNAs were labeled using the miRCURY™ Array Power Labeling kit (Exiqon). miRNA array hybridization was performed, and unbound miRNA labels were washed away using the miRCURY™ Array wash buffer kit (Exiqon). The arrays were scanned on an Axon 4000B scanner (Molecular Devices), and the signal intensity was determined using GenePix Pro 6.0 software (Molecular Devices). miRNAs with a 1.2-fold increase or decrease in expression were regarded as differential expression.

RNA of sufficient quality from eight group samples (n = 3, total 24 samples) was submitted to KangChen-Biotech (Shanghai, China). RNA groups above mentioned by applying the miRCURY locked nucleic acid (LNA) microarray platform (Exiqon, Denmark). All procedures were carried out according to manufacturer’s protocol. Briefly, 1 μg total RNA was labeled with the Hy3™ or Hy5™ fluorophores, using miRCURY™ Array Power Labeling kit (Exiqon, Denmark). After stopping the labeling procedure, briefly spin the reaction and leave it at 4 °C. The two samples from the Hy3™ and Hy5™ labeling reactions are combined on ice. The samples were hybridized on a hybridization station using miRCURY™ LNA miRNA array (v.18.0) containing Tm-normalized probes for 847 human miRNAs. Microarrays with labeled samples were hybridized at 56 °C for overnight using a heat-shrunk hybridization bag and washed using miRCURY Array Wash buffer kit (Exiqon, Denmark). After hybridization, the chip slides were washed, dried, and scanned immediately. Each miRNA spot was replicated for four times on the same slide and two microarray chips have been used for each group.

The labeled RNAs and miRCURY Array chip (Shanghai Kangcheng BioEngineering Co., Ltd., Shanghai, China) were hybridized in accordance with the manufacturer’s instructions. Briefly, 180 μl reaction mixture (25 μl labeled miRNA, 90 μl 2X hybridization buffer and 65 μl nuclease-free buffer) was incubated at 95°C in the dark for 2 min and subsequently placed in an ice bath for 10 min. At the same time, the miRCURY Array chip was assembled according to the manufacturer’s instructions (Shanghai Kangcheng BioEngineering Co., Ltd.). The reaction mixture was loaded through the loading port and the 1X hybridization buffer was used to fill the hybridization chamber. The microarray chip was packaged in a protective bag and placed vertically into water (95°C) for 12 h, followed by drying in an oven at 56°C over night. The chip was washed using the miRCURY Array Wash Buffer kit (cat no. 208021, Exiqon, Inc., Woburn, MA, USA) according to the manufacturer’s instructions, followed by centrifugation at 200 × g for 5 min to dry the chip. The scanning procedure was performed using an Axon GenePix 4000B microarray scanner (Axon Instruments, Inc., Foster City, CA, USA) to obtain the scanning profiles, followed by data analysis and analysis of the significant difference using GenePix Pro6.0 software (Axon Instruments, Inc.).