Dec 03, 2024

Public workspacesiRNA-mediated Knockdown – RAW 264.7

The Journal of Cell Biology
DOI
https://dx.doi.org/10.17504/protocols.io.4r3l29owjv1y/v1
  • Amanda Bentley-DeSousa1,2,3,4,5,
  • Shawn Ferguson1,2,3,4,5,6
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Amanda Bentley-DeSousa
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l29owjv1y/v1
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. siRNA-mediated Knockdown – RAW 264.7. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29owjv1y/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 02, 2024
Last Modified: December 03, 2024
Protocol Integer ID: 113419
Keywords: RAW264.7, siRNA knockdown, knockdowns in raw, mediated knockdown, raw
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to perform siRNA-mediated knockdowns in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
Lipofectamine RNAiMAX (Invitrogen, 2448190)
Opti-Mem (Thermo Fisher Scientific, 31985062)
Troubleshooting
Day 0
Plate 2.5 x 10^5 RAW 264.7 cells per well in a 6-well dish.
Day 1
30m
Transfect cells using Lipofectamine RNAiMAX and gene specific siRNAs.
RNAiMAX protocol
In Tube 1 combine Amount112.5 µL Opti-MEM and Amount4.5 µL Lipofectamine RNAiMAX then incubate for Duration00:05:00 . In Tube 2 combine Amount132 µL Opti-MEM and Amount2 µL siRNA (final concentration of 100 nM) then incubate for Duration00:05:00 . Combine Tube 1 and Tube 2. Incubate for Duration00:20:00 . Add drop-wise to 1 well containing Amount1.75 mL of media.

30m
Day 3
Change the media and perform subsequent experiments.