2.3. Target-triggered universal probe hybridization strategy for the detection of multiple miRNAs

Depending on experiment scale, an appropriate amount of capture probe-conjugated microspheres was removed from storage and transferred to 96-well plates in the amount of 0.5 μL stock/well. After magnetic separation, the supernatant was discarded. Mixture of miRNA targets and reporter probes prepared in designated concentration were added into each well (25 μL/well, in buffer A), and incubated at the designated temperature for 1 h. In a duplex assay, the two microsphere types were pooled at an equal volume before dispensing to the 96-well plate. After that, the microspheres were washed once with 50 μL wash buffer. The detection step was performed in 50 μL/well of SA-PE prepared in buffer A containing 1% BSA. After 30 min of incubation, the microspheres were washed once in 100 μL wash buffer, and then re-suspended in 100 μL of sheath fluid for analyzing on the Luminex 200 instrument.