Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Total RNA was isolated using ISOGEN II (311-07361, Nippon Gene). The medium was removed, 0.5 mL of ISOGEN II was added to the culture plate, and cell lysates were obtained by pipetting. After adding 0.2 mL of RNase-free water and mixing vigorously, the mixture was allowed to stand at 23–28 °C for 15 min. After centrifugation at 12,000×g for 15 min, the supernatant was recovered. An equal volume of isopropanol was added and the mixture was allowed to stand at room temperature for 10 min, followed by centrifugation at 12,000×g for 10 min to obtain a precipitate. The precipitate was washed twice with 80% ethanol before dissolving in RNase-free water as the total RNA. The isolated total RNA was reverse-transcribed using a PrimeScrip 1st strand cDNA Synthesis Kit (6110A, TaKaRa). The reverse transcription reaction was performed in a thermal cycler at 42 °C for 60 min, 95 °C for 5 min, and 4 °C for 5 min. After this reaction, cDNA was amplified using a TaKaRa PCR Thermal Cycler Dice (TP 650, TaKaRa) using a PCR Amplification Kit (R011, TaKaRa). Finally, the PCR products were separated on a 3% agarose gel and visualized by staining with ethidium bromide. For qPCR, cDNA was synthesized using a PrimeScript RT reagent Kit (RR037A, TaKaRa). qPCR was performed using TaKaRa SYBR Premix Ex Ta II (Tli RNaseH Plus) (RR420A, TaKaRa) and the Thermal Cycler Dice qPCR apparatus integrated with Real Time System Software (TaKaRa – TP800). The primer sets used for amplification are listed in Table ^S2.