2.6. In vitro delivery of BERA/GFP-siRNA in human carcinoma Cells.

SK-Hep1-Luc-GFP or A549-Luc-GFP cells (5 × 10⁴ cells/well) were seeded in 12-well plate and grown overnight. BERA/GFP-siRNA-loaded LPP nanocomplex was added into each well to a final concentration of 5 nM. LP3000 and IVJ-PEI formulations were included for comparison. After 72 h of treatment, cells were collected and total RNA was extracted with Trizol and Direct-zol RNA MiniPrep Kit (Zymo Research). cDNA was synthesized from 500 ng total RNA using NxGen M-MuLV reverse transcriptase (Lucigen, Middleton, WI, USA), with random hexamers or specific stem-loop primer for GFP-siRNA (5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG GGC AC-3′). Levels of GFP-siRNA and precursor GFP-siRNA were determined by using U6 as the internal standard. Levels of GFP-mRNA were determined by using 18S as the internal standard. A forward primer 5′-GCG CGC AGT TGT ACT CCA GCT T-3′ and reverse primer 5′-GTG CAG GGT CCG AGG T-3′ were used for real-time qPCR analysis of GFP-siRNA. The forward and reverse primers for qPCR analysis of GFP mRNA were 5′-ACG TAA ACG GCC ACA AGT TC-3′ and 5′-AAG TCG TGC TGC TTC ATG TG-3′, respectively. The forward and reverse primers for 18S were 5′-GTA ACC CGT TGA ACC CCA TT-3′ and 5′-CCA TCC AAT CGG TAG TAG CG-3′, respectively. The forward and reverse sequences for U6 were 5′-CTC GCT TCG GCA GCA CA-3′ and 5′-AAC GCT TCA CGA ATT TGC GT-3′, respectively. All real-time qPCR experiments were performed using iTaq Universal SYBR Green Supermix on a CFX96 Touch real-time PCR system (Bio-Rad, Hercules, CA, USA). Cells were treated in triplicate and assayed separately. The comparative threshold cycle (Ct) approach with the formula 2^−ΔΔCt was utilized to calculate the relative gene expression.

Live cell GFP fluorescence images were acquired with an Olympus IX81 microscope (Olympus, Center Valley, PA, USA) at 72 h post-transfection. To quantify the GFP fluorescence intensity, cells were first lysed with 1% Triton dissolved in PBS (1 mM EDTA). A 100 μL aliquot was collected for the measurement of GFP fluorescence (Excitation (Ex)/Emission (Em) = 488 nm/509 nm) on a microplate reader (SpectraMax M2, Molecular Devices, USA). The fluorescence intensity was normalized to total protein concentration in corresponding sample, which was quantitated by using BCA assay kit.