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RNA interference

SC Shawn C. Chafe
SD Shoukat Dedhar

Last updated date: Feb 24, 2022 Views: 644 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Aug, 2021
Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors
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siRNA Transfection with siLentFect (6-well)


Required reagents:

  • siLentfect (Bio-Rad #1703360)
  • siRNA to gene of interest (20 mM – Qiagen)
  • OPTI-MEM (Thermofisher Scientific #31985070)
  • 6-well plates (VWR # CA62406-161)

 

Protocol:

  1. siRNA should be 20 μM
  2. Label tubes and plates. Let reagents and OPTI get to RT before use. Vortex reagents.
  3. Add 7.5 μL siRNA and 500 μL Opti-MEM. Vortex.
  4. To another tube: add 2.5 μL siLentfect and 500 μL Opti. Vortex.
  5. Combine 3 and 4 for 20 minutes.
  6. Meanwhile, split cells so that they will be ~75% confluent at plating (Cell type dependent and requires titration to determine this value. 5x104 cells/cm2 is a good starting point).
  7. Add 1 mL of cells at desired concentration (see #6) to each well of 6-well. Add the combined siRNA mix and pipette up and down.
  8. Change media or split the next day. 
  9. Assay efficiency of gene depletion 72 hours post-transfection.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Chafe, S and Dedhar, S(2022). RNA interference. Bio-protocol Preprint. bio-protocol.org/prep1549.
  2. Chafe, S. C., Vizeacoumar, F. S., Venkateswaran, G., Nemirovsky, O., Awrey, S., Brown, W. S., McDonald, P. C., Carta, F., Metcalfe, A., Karasinska, J. M., Huang, L., Muthuswamy, S. K., Schaeffer, D. F., Renouf, D. J., Supuran, C. T., Vizeacoumar, F. J. and Dedhar, S.(2021). Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors . Science Advances 7(35). DOI: 10.1126/sciadv.abj0364

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