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 (1.5×107) are resuspended to 3×107 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.
