Thiscomplex initiates the RNA synthesis.
PCR template method for dsRNA Synthesis
Total RNA was isolated from a diatom colony using the TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacturer’s protocol. The RNA fraction was treated with DNase I (Takara, Otsu, Japan). Double-stranded cDNA was synthesized from 2 μg of total RNA with random primers (9-mers) using a PrimeScript Double Strand cDNA Synthesis Kit (Takara). The resultant cDNA was quantified using a Qubit dsDNA HS Kit.
The dsRNA can be made from cDNA or genomic DNA ..
DsRNA was purified as described by Okada et al. with a few modifications (, ). Briefly, the microbial sample was disrupted in liquid nitrogen in a mortar and total nucleic acids were manually extracted. DsRNA was purified twice through a micro-spin column (empty Bio-spin column; Bio-Rad Laboratories, Inc., Hercules, CA, USA) containing cellulose powder (Cellulose D; ADVANTEC, Tokyo, Japan) to obtain pure dsRNA. The dsRNA eluted from cellulose powder in MQ water was treated with DNaseI (amplification grade, Invitrogen, Carlsbad, CA, USA) and S1 nuclease (Invitrogen) in nuclease buffer (57 mM CH3COONa, 9.5 mM MgCl2, 1.9 mM ZnSO4, and 189 mM NaCl) and was then incubated at 37°C for 2 h. The final concentrations of CH3COONa, MgCl2, ZnSO4, and NaCl were adjusted to 90 mM, 15 mM, 3 mM, and 300 mM, respectively. DsRNA was purified using an RNeasy Mini Kit (Qiagen, Valencia, CA). A one-tenth volume of 10 × ShortCut buffer and 10 × MnCl provided with ShortCut RNase III (NEB Japan, Tokyo, Japan) was added to the dsRNA solution and fragmented by ultrasound at 4°C in Snap-Cap microTUBEs using a Covaris S220 (Woburn, MA, USA). The fragmentation conditions were as follows; run time 35 s, peak power 140.0 W, duty factor 2.0%, and 200 cycles/burst. Fragmented dsRNA was divided into two equal volumes, and maintained at 37°C with or without ShortCut RNase III (NEB). DsRNAs were then purified using a ZymoClean Gel RNA Recovery Kit (ZymoResearch, Orange, CA). Note that dsRNA purification from M. oryzae was carried out in the laboratory of Prof. Teraoka.