Protocol
For transfecting T-cells, we have found that electroporation can be a very efficient way to get siRNAs in. The plus is that high siRNA transfections can be seen (70-95%), but the downside is that a lot of siRNA and cells have to be used. As you might expect, the high voltages can kill most of the cells. We typically make 250 pmol/μl siRNA stock, and then add 10 μl per cuvette. Therefore, we get ~ 25 transfections/0.2 μmol scale RNA synthesis.
For electroporations, 1-10 μl of a 250 pmol/μl siRNA soln and/or 20 ug of a GFP plasmid are added to pre-chilled 0.4 cm electrode gap cuvettes (Bio-Rad, Hercules, CA).
Cells (1.5x107) are resuspended to 3x107 c/ml in cold, serum-free RPMI, added to the cuvettes, mixed, and pulsed once at 250-450 mV (the voltage has to be optimized for cell type and the individual electroporator), 960 uF, 200 ohms with a Gene Pulser electroporator (BioRad, Hercules, CA). Cells are plated into 6-well culture plates, each well containing 8 ml complete medium, and incubated at 37°C in a humidified 5% CO2 chamber. Cell viability
immediately after electroporation is typically around 40-60%, or even less.
The figure to the left is flow cytometric analysis of CD8 silencing in a T-cell line. The cell doubling time for this particular cell line (mouse E10) is approx. 12 hours. When CD8α siRNAs are co-transfected with the GFP reporter
vector, CD8α expression, but not GFP expression, is markedly reduced. Several cell populations are evident, with the major CD8α silenced population displaying approximately 5- fold reduced CD8α expression. The majority of cells within this population do not express GFP.
However, every cell that does express GFP also silences CD8α. Therefore inclusion of a reporter vector will be a great way to enrich for silenced cells using FACS. The time course at the right illustrates several important points:
1. siRNA silencing is transient 2. silencing is not 100% penetrant 3. siRNAs does not appear to be toxic to the cell (compare the kinetics of the fates of GFP alone vs GFP+siRNA transfected cells).
An additional note that I should make is that not all siRNAs are going to work. At the moment, it is not clear what rules govern siRNA effectiveness.
If you have any questions, do not hesitate to contact me by email- I would also like to hear any improvements made to this protocol. Michael McManus
