Quantification of Ku70 Gene Expression

Total RNA extraction was conducted using TRIzol (Invitrogen, Carlsbad, CA, USA) based on manufacturers' specifications. Using the M-MLV RT kit (Promega, Madison, WI, USA), 2 μg RNA was reverse-transcribed into cDNA. The PCR reaction using resultant cDNA was conducted using the following primers: Ku70: 5′-GTGGTCACACACGAGCTTATT-3′ (sense) and 5′-CAAATGTCTGATGTTGGTGAACC-3′ (antisense) and β-actin: 5′-TGGATCAGCAAGCAGGAGTA-3′ (sense) and 5′-TCGGCCACATTGTGAACTTT-3′ (antisense). The thermal cycle for PCR was set as follows: 95°C for 2 min, 30 cycles of 94°C for 30 s, 56°C for 45 s, and 72°C for 45 s, with a final extension at 72°C for 7 min on a Lightcycler 480 system (Applied Biosystems, Foster City, CA, USA) using the SYBR Green Master Mixes (Takara, Japan). The Ku70 expression level was normalized to β-actin followed by the quantification through the 2^−ΔΔCt method [15 (link)].