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Reverse Transfection of Adherent Cells with siRNA in 384-Well Plates

Berkeley Screening Center

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Reverse Transfection of Adherent Cells with siRNA in 384-Well Plates

This modified protocol from Qiagen is provided as a starting point for optimization of siRNA transfection of adherent cells in a single well of a 384-well plate using HiPerFect Transfection Reagent.  In this protocol, cell plating and transfection are performed on the same day.

Important points before starting

  • Cells should be in optimal physiological condition at the time of transfection. The amount of cells seeded depends on the cell type and time of analysis.
  • If siRNA has been ordered from QIAGEN, it is delivered lyophilized and must be resuspended prior to transfection. To resuspend, follow the instructions provided with the siRNA. We recommend starting your assay development with 500 nMolar siRNA to mimic the siRNA libraries you will use for screening.

  • Nucleic acid amounts in the protocol refer to siRNA.


1. Spot 5 μl of 500 nMolar siRNA suspended in Buffer/RNase-free water into a single well of a 384-well plate.

  • The use of this 500 nMolar concentrated working solution will give a final siRNA concentration of 50 nM after addition of cells to complexes in step 4).
  • Note: After this step, siRNA may be stored at -20°C for long time periods. siRNA may be stored in solution or may be dried in the plate at room temperature (15-25°C) before storage.

2. Add 0.5 μl of HiPerFect Transfection Reagent to 9.5 μl of culture medium without serum. Add this Transfection Mix to the prespotted siRNA.

  • Note: To ensure accurate pipetting, diluted HiPerFect Reagent should be prepared in a larger volume for use in multiple wells.
  • IMPORTANT: The amount of HiPerFect Transfection Reagent and siRNA required for optimal performance may vary, depending on the cell line and gene target.

3. Incubate for 5-10 min at room temperature (15-25°C) to allow formation of transfection complexes.

4. Seed 4000-10,000 cells in 30 μl of an appropriate culture medium (containing serum and antibiotics) into the well, on top of the siRNA-HiPerFect Reagent transfection complexes.

5. Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing or phenotype after an appropriate time (e.g., 6-72 h after transfection, depending on experimental setup). Change the medium as required.

  • Note: The optimal incubation time for gene silencing analysis depends on cell type, the gene targeted, and the method of analysis. This can be determined by performing a time-course experiment.

 Example of different conditions using BSC siRNA library stocks.

Stock siRNA concentration (nM) 500 500 500 
Stock siRNA volume (uL) 33 33  33 
Number of replicates 2
Final siRNA Concentration on cells (nM) 100 50 10
siRNA stock use per assay plate 10 5 1
Transfection mix (uL) 10 10 10
Cell volume (uL) 30 35 39
Total volume in well (uL) 50 50 50
siRNA aliquot left (uL) 13 23 31
Enough for duplicate hit confirmation? no yes yes










Last Updated on Wednesday, 14 January 2009 11:40  

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