Total RNA is extracted from plants using TRIzol reagent according to the supplier's instructions (Takara, Japan, or equivalent). For homogenizing plant material, plants and four steel balls are placed in a 2-ml Eppendorf tube and homogenized using a Shake Master version 1.2 (BMS-12). Alternatively, RNA can be extracted from plants using the ATA method. However, because ATA inhibits the activity of reverse transcriptase, the RNA cannot be used for real-time polymerase chain reaction (RT-PCR) and cDNA synthesis. For a detailed protocol of this method, see Nakashima and Yamaguchi-Shinozaki (2002).
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