Isolation and Analysis of mRNA from Tissues

Fresh isolated tissues from WT and Top3β^−/− mice were treated with Trizol reagent, mixed with 200 µL chloroform, and centrifuged at 4 °C for 15 min at 8000× g. The supernatant was recovered, mixed with 500 µL 2-propanol, and centrifuged at 4 °C for 10 min at 8000× g. The mRNA pellet was extracted with 75% ethanol to increase RNA purity. After centrifugation, ethanol was removed by evaporation and the pellet was dissolved in diethylpyrocarbonate-treated distilled water (Tech & Innovation, Chuncheon, South Korea). A reverse-transcription aid kit (Thermo Scientific, Foster City, CA, USA) was used to synthesize cDNA, which was then subjected to q-PCR using SYBR Green PCR Master Mix (Roche, Basel, Switzerland) and a LightCycler 480 Instrument II (Roche). GAPDH was used as an internal control. Sequences and sources of all primers used for PCR are listed in Supplementary Table S1 [16 (link),62 (link),63 (link),64 (link),65 (link)]. The Spo11 TaqMan probe was used.