Quantitative Real-Time PCR Protocol

Total RNA was isolated by using a total RNA kit (TIAGEN, Beijing, China) following the manufacturer’s instructions, followed by [88 (link),89 (link)]. For the complementary DNA (cDNA) synthesis, 2 mg of total RNA was reverse-transcribed using a Prime-Script TM II First-Strand cDNA synthesis Kit (TakaRa, Dalian, China). The quantitative real-time PCR (qRT-PCR) analysis was performed with a Light Cycle 480 II system (Roche, Basel, Switzerland) using an SYNR Premix Ex TaqTM kit (TakaRa, Dalian, China). The UBI-eq gene was used as an internal control. The primer sequences used for the PCR are given in (Supplementary File S5, sheet 2). Three replicates were made for three separate RNA extracts from the three samples.