Extract total RNA from at least 2 x 10^6 cells using TRIZOL reagent . #####%%%%% output "total RNA" are used in line4
{"action": "extract", "output": "total RNA", "reagent": ["TRIZOL reagent"], "volume": ["at least 2 x 10^6 cells"], "container": [""]}
Reverse-transcribe 60 ng of total RNA with a High-capacity cDNA Reverse Transcription kit. #####%%%%% output "cDNA" are used in line6
{"action": "reverse-transcribe", "output": "cDNA", "reagent": ["total RNA"]}
Prepare a reaction mix with 12.5 μl 2X Sybr Green PCR Master Mix, 2.5 μl 1 μM primer mix, 2 μl cDNA (5 ng/μl), . #####%%%%% output "cDNA" are used in line8
{"action": "prepare", "output": "cDNA", "reagent": ["2X Sybr Green PCR Master Mix", "primer mix", "cDNA"]}
Perform Real-Time PCR on 10 ng of cDNA using a Power Sybr Green PCR Master Mix with the following protocol: 50°C (2 min), 95°C (10 min), 95°C (15 sec), 60°C (1 min), for 40 cycles.
{"action": "perform", "output": "", "device": ["Real-Time PCR"], "reagent": ["cDNA"], "volume": ["10 ng"]}
Conduct a dissociation assay to evaluate primer specificity.
{"action": "conduct", "output": ""}
Analyze data with built-in SDS Analysis Software .
{"action": "analyze", "output": "", "device": ["built-in SDS Analysis Software"]}
adjust conditions if issues are detected before repeating the assay
{"action": "adjust", "output": ""}