Quantitative Real-Time PCR Protocol

Total RNA was extracted using Trizol reagent (Invitrogen, Cat. 15596026, Waltham, MA, USA) and digested with RNase-free DNase I (TaKaRa, Dalian, China), and 2 μg of total RNA was used for the first-strand cDNA synthesis (TaKaRaPrimeScript™ II 1st strand cDNA synthesis kit, Code No. 6210A) based on the manufacturer’s protocol. QRT-PCR was performed with ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Waltham, MA, USA) using the TB green™ premix Ex Taq™Ⅱ kit (Takara, Dalian, China). The total volume of 20 μL reaction mixture consisted of 10 μL 2 × TB Green Premix Ex Taq Ⅱ (ROX plus), 0.8 μL gene-specific forward and reverse primers (10 μM each), 1 μL cDNA template, and 7.4 μL RNase-free water. Reaction conditions were 95 °C for 5 min followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C. Planarian elongation factor 2 (Djef2) was utilized as the reference gene in all of the experiments [32 (link)]. Expression ratios were determined with the 2^−ΔΔCT method, which was described by Livak and Schmittgen [33 (link)]. The primers used in this work were listed in Supplementary Excel File S1.