Prepare annealing solution of 50 µM RNA/DNA oligos with 50 mM NaCl in DNase/RNase-free water, aliquot 50 µl in PCR tube, . #####%%%%% output "inhibitor compound" are used in line4
{"action": "prepare", "output": "inhibitor compound", "concentration": ["50 \u00b5M RNA/DNA oligos"], "reagent": ["DNase/[RNase-free] water"], "volume": ["50 µl"], "container": ["PCR tube"]}
Dissolve inhibitor compound in DMSO to 10 mM, if needed, prepare serial dilutions in Milli-Q water. #####%%%%% output "water" are used in line6
{"action": "dissolve", "output": "water", "reagent": ["inhibitor compound"]}
Add water (20 µl in blanks, 10 µl in controls) to 96-well plate.
{"action": "add", "output": "", "reagent": ["water"], "container": ["96-well plate"]}
Add 80 µl RT reaction mix (1.25x).
{"action": "add", "output": "", "volume": ["80 \u00b5l RT reaction mix (1.25x)"]}
Add 10 µl inhibitor dilution to samples, to each well. #####%%%%% output "EDTA" are used in line12
{"action": "add", "output": "EDTA", "volume": ["10 \u00b5l inhibitor dilution"], "container": ["samples"]}
Stop reaction with 50 µl EDTA (0.5 M, pH 8.0).
{"action": "stop", "output": "", "reagent": ["EDTA"], "volume": ["50 µl"]}
Quantify reaction with Victor 3 at 490/528 nm, , report inhibitor values as percentage of control. #####%%%%% output "490/528 nm" are used in line16
{"action": "quantify", "output": "490/528 nm", "device": ["Victor 3"]}
subtract blank value from samples #####%%%%% output "blank value" are not used
{"action": "subtract", "output": "blank value", "reagent": ["490/528 nm"]}
Calculate IC50 value as the concentration reporting 50% reduction of signal compared to control. #####%%%%% output "signal" are not used
{"action": "calculate", "output": "signal", "concentration": ["IC50 value"]}