LNA Library Screening for Cancer Cell Viability

The screening of the LNA library (Exiqon, Woburn, MA, USA) was performed as previously described [16 (link)]. Briefly, SW480 cells (2 × 10³ cells/well) were seeded in 96-well plates. DharmaFECT 2 Transfection Reagent (Dharmacon, Lafayette, CO, USA) was diluted in Optimem medium (Thermo Fisher Scientific, Waltham, MA, USA), seeded on LNAs and incubated for 15–20 min at room temperature. The transfection mixture was then added to the cells at the final LNA concentration of 25 nM. These conditions were formerly set up to ensure ~90% transfection efficiency. The percentage of transfected cells was monitored with a control siRNA against NF1b mRNA (40 nM), and verified by Real-Time PCR. As a positive control, the siRNA against PLK-1 was added on empty wells of the library plates. Cancer cells were assayed 72 h post-transfection with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) on a Beckman DTX 880 plate luminometer (Beckman Coulter, Brea, CA, USA). The relative viability was calculated and normalized to the average of all samples, excluding positive controls. The experiment was performed in triplicate. Values below and above twofold the standard deviation were considered significant and were used in further analyses.