DNA isolation from human retina

DNA isolation from mitochondrial and nuclear pellets was performed using the protocol available in Qiagen QIAamp DNA Mini Kit (Qiagen, Valencia, CA) with several modifications. To prevent the artifactual formation of 8-oxo-dG during the DNA isolation, 8-HQ as an antioxidant¹³ was added to all the extraction buffers to a final concentration of 0.35 mM. Briefly, 200 μL of ATL buffer was added to the pellet and proteinase K (10 μL of 20 mg/mL solution) was added and incubated at 56 °C for 1 h with gentle shaking. After the incubation, 4 μL of RNase A solution (100 mg/mL) was added, and the sample was incubated at room temperature for 2 min, followed by the addition of 200 μL of AL buffer and incubating at 70 °C for 10 min. The sample was then mixed with 200 μL EtOH, and the mixture was loaded onto a Mini spin column and centrifuged at 6,000 g for 1 min. The column was washed with 500 μL of AW1 buffer and 500 μL of AW2 buffer sequentially, and the DNA was finally eluted with 400 μL of nuclease free H₂O.