siRNA Delivery Success – Assessing Target Knockdown & Cell Viability
- How do you measure siRNA delivery success?
- Do you only look at target knockdown?
- Do you also look at cell viability?
Why assess cell viability in addition to assessing mRNA or protein reduction, you may ask? What if you observed significant target silencing after siRNA delivery, but a large number of cells died during transfection? Without measuring cell viability you might be led to believe that siRNA was successfully delivered to the cells, when in fact, the observed target knockdown was due to non-specific cell death associated with sub-optimal transfection conditions.
To demonstrate this point, consider the data from a transfection optimization experiment (Figure 1) which reveals the effect of GAPD siRNA delivery parameters on cell viability and GAPD mRNA levels in LNCaP (A) and HEK293 (B) cells. For both cell lines, at low (5x103 cells per well), medium (10 x103 cells per well) and high (25 x103 cells per well) densities, cell viability (l) is compromised with increasing lipid volumes. The greatest effect is observed at low cell densities and high lipid volumes (pink boxes), where both cell viability and GAPD mRNA levels are low. By assessing only mRNA levels, one would be misled to believe that good target knockdown had been achieved. However, analyzing the mRNA levels in parallel with the cell viability data demonstrates that the low mRNA levels are purely a function of low cell viability.
Further analysis of Figure 1 shows that both good cell viability and target knockdown can be achieved by using a transfection optimization experiment to identify the best combination of transfection parameters. For example, using 75% as the minimum level for cell viability and more than 75% reduction in mRNA expression (blue bars, normalized values), acceptable viability and good knockdown can be achieved at high cell densities (green boxes). Under these conditions, cell viability is preserved, thus the target knockdown observed is truly siRNA-mediated. It is noteworthy that at low cell densities, a small window of optimal conditions can also be utilized (i.e., for LNCap, 5 x103 cells transfected with 0.05-0.1 uL of DharmaFECT 3).
Clearly target knockdown is not sufficient to verify siRNA delivery success. Delivery efficiency should be measured by the number
of viable cells transfected in conjunction with assays that measure siRNA induced gene silencing. Delivery success varies with delivery conditions and is very cell line dependent, thus optimization is paramount. In conclusion, when evaluating siRNA delivery it is prudent to assess both cell viability and target mRNA levels for the most accurate measure of true siRNA-mediated knockdown.
