Profiling MicroRNA Expression Using MicroRNA Assay

Protocol Overview

A 1:500 dilution of the miRNA Assay Controls is prepared, followed by an annealing master mix (by combining 13 μL of Annealing Buffer, 26 μL of nCounter miRNA Tag Reagent and 6.5 μL of the 1:500 miRNA Assay Controls dilution). 3.5 μL of the annealing master mix is then aliquoted into each tube of a 12 x 0.2 mL strip tube, and 3 μL maximum of RNA sample is added to each tube. The strip is placed in a thermal cycler and the Annealing Protocol initiated. Next, 19.5 μL PEG and 13 μL Ligation Buffer is combined to prepare a ligation master mix. Following completion of the Annealing Protocol, when the thermal cycler has reached 48°C, 2.5 μL of the ligation master mix is added to each tube, and the tubes incubated at 48°C for 5 min. The thermal cycler is then opened and the caps carefully removed from tubes, leaving the strip in place in the heat block, and 1.0 μL of Ligase is added directly to the bottom of each tube while incubating at 48°C. Immediately after addition of Ligase to the final tube, tubes are recapped and the Ligation Protocol is initiated. After completion of the Ligation Protocol, 1 μL of Ligation Clean-Up Enzyme is added to each reaction. The tubes are then returned to thermal cycler and the Purification Protocol initiated. Following completion of this protocol, 40 μL DEPC (or RNAse-free) H2O is then added to each sample. A master mix is then created containing 130 μL of the Reporter CodeSet and 130 μL of hybridization buffer by adding the hybridization buffer to the tube containing the Reporter CodeSet. 20 μL of master mix is added to each of 12 tubes in a tsrip tube. Samples from the miRNA sample prep protocol are then denatured at 85°C for 5 minutes and quick-cooled on ice. A 5 μL aliquot from the miRNA Sample Preparation Protocol is then added, followed by 5 μL of Capture ProbeSet to each tube. These are immediately placed in the 65°C thermal cycler and incubated for at least 12 hours. Once removed from the thermal cycler, the user can then proceed immediately to post-hybridization processing with the nCounter Analysis System User Manual (MAN-C0035) or the nCounter SPRINT Profiler User Manual (MAN-10017).