External ID: TargetID_659_CEMA

Protocol: Black, standard capacity streptavidin-coated 384-well plates (Pierce 15407) were first washed with 50 L of phosphate buffer (100 mM, pH 7.0; PB7) three times using a Biotek 405 ELX plate washer. Subsequently, 5 L of biotinylated pre-miRNA substrate (500 nM final) was dispensed into the plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific). Plates were then centrifuged for 1 min at 1,000 RPM (223 g), sealed with plate tape, and incubated overnight at 4 C. The following morning, plates were washed three times with 50 L of PB7, followed by the addition of 5 L of Dicer digest buffer (20 mM Tris, 12 mM NaCl, 2.5 mM MgCl2, 1 mM fresh DTT, and 4.5% DMSO) and centrifugation. Compounds (50 nL of 5 mM DMSO stock, 25 M final) were then added into the sample wells using a Sciclone (Caliper) liquid handler with V&P pintool; the same volume of DMSO was added to the control wells. The plates were incubated at 25 C for 15 min before addition of 5 L of digest buffer containing 217 g/nL Dicer (108 g/mL Dicer, 5% glycerol and 0.01% Triton X-100 final, excess with respect to pre-miRNA). For the positive control wells, digest buffer without Dicer was added. The plates were centrifuged again and resealed before being placed in a 37 C incubator for 5 h. After Dicer cleavage, plates were washed three times with 50 L of PB7. mTet-HRP in PB7 (10 L, 750 nM final) was then dispensed into each well. The plates were subsequently centrifuged, sealed, and incubated at 25 C for 2 h. Plates were then washed three times with 50 L of wash buffer (2 mM imidazole, 260 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20, pH 7.0), followed by washing three additional times with 50 L of PB7. Finally, SuperSignal West Pico (25 L; Pierce) was added, the plates were incubated at 25 C for 5 min, and chemiluminescence signal was detected using a PHERAstar plate reader using LUM plus module (BMG Labtech).

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.

For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.