Nucleofection™ of siRNA Libraries
High-throughput transfection of siRNA libraries has become an indispensable tool in target identification and validation. However, meaningful siRNA screening results strongly depend on efficient delivery of siRNA into the selected cell type. Most screenings have so far been limited to easy-to-transfect adherent cell lines. The high-throughput Nucleofection™ Platforms now enable these approaches in more relevant cell types - primary and hard-to-transfect cells:
Example: siRNA library screens in hard-to-transfect HUVEC and Jurkat cells
In collaboration with Thermo Fisher Scientific (Dharmacon Products), Lonza performed two different siRNA library screens using the 96-well Shuttle™ System. The setup and results of both screens are summarized in our joint White Paper.
- Primary human umbilical vein endothelial cells (HUVEC) were screened with a combined protein kinase and a cell-cycle library to identify targets that affect cell viability.
- Jurkat T cells were screened with an apoptosis library to identify genes that regulate FAS-mediated cell death (see figure below).
FAS-induced apoptosis in Jurkat Cells - Reproducible hits from three screens. Jurkat cells (clone E6-1) were transfected in three independent experiments with the Dharmacon Human siARRAY ON-TARGETplus siRNA Library for Apoptosis (targeting 558 genes). Apoptosis was induced by adding 10 ng FAS-L to the cells 48 h post transfection. Cell viability was analyzed after 2h. The mean of robust Z-scores of cell viability measures was calculated for three independent experiments. Targets with an |MAD| of at least 5 are marked as potential hits.