Silver: miRNA screens

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  • MiRNA are arrayed in 96-well plate and stored at –20C. Transfection is performed with siPORT-NeoFx reagent (Ambion #4510) in OPTI-MEM.
  • OPTI-MEM and NeoFX should be at room temperature at time of transfection.
  • For each transfection mix 0.5 microliter NeoFX with 29.5 µl OPTI-MEM. Incubate at room temperature for 10 minutes. Make master mix for large-scale transfection.
  • Add 30 microliter of NeoFX/OPTI-MEM mix to each well containing lyophilized miRNA. Incubate at room temperature for 10 minutes.
  • In the mean time, trypsinize cells and dilute them to ~ 85,000 cells/ml in DMEM/10% FBS/Pen-Strep (this will give you about 6000 cells per well at time of plating).
  • Add 70 µl of the cell suspension (equivalent to 6000 cells) to miRNA/NeoFX/OPTI-MEM mixture.
  • Mix and transfer to flat bottom 96-well Corning plate (#3614).
  • Allow transfection to proceed for 48 hours.
  • Screen cells using assay of choice.