Comprehensive RNA Extraction and Analysis

Total RNA was extracted using an RNA extraction kit (Invitrogen, China) and digested with DNase I (Takara, China) according to the manufacturers’ instructions. The RNA quality and integrity were analysed by agarose gel electrophoresis and the RNA concentration was determined using a biophotometer (METASH, B–500, China). cDNA was synthesized from total RNA using AMV Reverse Transcriptase (Promega, China). Small RNA was extracted using an RNAiso kit for small RNA (Takara, China) and digested with DNase I (Takara, China) according to the product manuals. Reverse transcription was performed with a cDNA Synthesis Kit (Promega, China) in combination with a stem-loop RT-PCR technique73 (link). Quantitative RT-PCR was performed on a 7500 RT-qPCR system (Applied Biosystems, USA) with SYBR Green Real-time PCR Master Mix (Toyobo, China) according to the manufacturer’s instructions. Gene expression was normalized to that of rice ACTIN1. Primers used for qRT-PCR are presented in Supplemental Table S1.