Hs actb 2 sg primers for β actin

To determine mRNA level knockdown efficiency of polyplexes, silencing of housekeeping gene GAPDH in H1299 and H460 was determined by real time PCR (RT-PCR). Cells were seeded at the density of 50,000 cells/well in 24-well plates for 24 h. bPEI or HAI-bPEI were formulated with 50 pmol of siRNA against GAPDH or scrambled siRNA at N/P = 5. Cells were transfected with polyplexes at a siRNA concentration of 125 nM for the first 4 h and 50 nM for an additional 20 h. Total mRNA was isolated from cells using the PureLink^® RNA mini kit (Life Technologies) following the manufacturer’s protocol in the addition of DNase I digestion (Sigma). The brilliant III ultra-fast SYBR^® green QRT-PCR master mix kit (Agilent Technologies, Santa Clara, CA, USA) was used to reverse transcribe 100 ng of mRNA to cDNA and perform RT-PCR. RT-PCR was conducted in a Stratagene Mx 3005p qPCR system (Agilent Technologies) and cycle threshold (Ct) values were exported from MxPro software (Agilent Technologies). Hs_GAPDH_2_SG primers for GAPDH and Hs_ACTB_2-SG primers for β-actin (Qiagen, Valencia, CA, USA) were used in this experiment. GAPDH gene expression was normalized to β-actin gene expression for quantification and comparison, and the untreated group represented 100% GAPDH expression. Experiments were conducted in triplicate.