Genejet reagent

For HR analysis we used the HR kit from Norgen Biotek Corporation (Canada), providing two different plasmids (dl-1 and dl-2) each containing a defective lacZ cassette that through an event of HR form a functional lacZ cassette. Briefly, 40 × 10⁴ A549 cells/well in 24-well plates were first transfected with SCAT7 targeting LNA GapmeRs following the protocol described earlier. Twenty-four hours post transfection, cells were transfected with 500 ng dl-1 plasmid and dl-2 plasmids together for HR assay, dl-1 and dl-2 alone for negative control, and a positive control plasmid, using GeneJet reagent (SignaGen Laboratories). Twenty-four hours after plasmid transfection, we induced DNA damage with 5 μM cisplatin for 24 h, and treated control cells with dimethyl sulfoxide (DMSO) only. Cells were harvested and DNA was isolated with QIAmp DNA Mini Kit (QIAGEN). The rate of HR was evaluated by RT-qPCR using the Assay Primer Mixture (detecting the recombined LacZ sequence) and Universal Primer Mixture (detecting the plasmid backbone) supplied in the kit. For RT-qPCR we used the following conditions: initial denaturation at 95°C for 3 min, then 95°C for 15 s, 61°C for 30 s and 72°C for 1 min repeated for 40 cycles.