2.9. Quantitative RT-PCR
GAPDH mRNA expression reduced by complex was checked using quantitative real-time PCR. HeLa cells were seeded into a 24-well plate at a density of 1.0×105 cells per well. After overnight incubation at 37°C in a 5% CO2 incubator, 160 μL complex with the final 200 nM siRNA at 20:1 N/P ratio was applied to the cells in Opti-MEM™ for 5 hours. As a positive control, Lipofectamine™ 2000 reagent was used according to the provided protocols. After incubation for 5 hours, the media were replaced with 500 μL of fresh supplemented DMEM and incubated further for 19 hours. After washing three times with nuclease-free water, the total RNA of the cells was purified using Tri-RNA reagent following the manufacturer’s protocol. The concentration and purity of purified RNA were measured using the Take3 plate as a nanodrop mode of a microplate reader (Epoch 2; BioTek Instruments, Inc., VT, USA). 100 ng of RNA was reversely transcribed to cDNA using ReverTra Ace® qPCR RT Master Mix with a gDNA Remover kit according to the manufacturer’s protocol. Used GAPDH-specific primer sequences were forward primer 5’-GTCTCCTCTGACTTCAACAGCG-3’ and reverse primer: 5’-ACCACCCTGTTGCTGTAGCCAA-3’. After mixing 10 ng of cDNA, primers and THUNDERBIRD® SYBR® qPCR Mix according to the manufacturer’s protocol, PCR reaction was performed following to the three-step cycle (Pre-denaturation; 95°C for 60 seconds, Denaturation; 95°C for 15 seconds, Annealing; 55°C for 30 seconds, Extension; 72°C for 60 seconds). GAPDH mRNA expression was analyzed using the QuantStudio 3 real-time PCR system (Applied Biosystems, CA, USA). The GAPDH mRNA expression was normalized by β-actin mRNA expression. The relative expression level was calculated using the ΔΔCt method.