Efficient siRNA Delivery into CLL Cells

Electroporation of siRNA into CLL cells was performed using Amaxa Human B-cell Nucleofection Kit (Amaxa, Cologne, Germany) as previously described.^{45 (link)} Briefly, 1 × 10⁷ PBMCs were mixed with 100 μl of Amaxa B-cell nucleofector solution, and 2 μg of siRNA was nucleofected using program X-05. This resulted in transfection efficiency of 30–60% (as determined with 2 μg pMaxGFP plasmid) and viability of 50–80% cells at 24 h. The following siRNA sequences were used (sense strand): Cdt1, 5′-GCAAUGUUGGCCAGAUCAA-3′ p27, 5′-GCAACCGACGAUUCUUCUACUCA-3′ p21, 5′-CUGUACUGUUCUGUGUCUU-3' (all from Dharmacon, Lafayette, CO, USA).