Non-coding RNA tiling array in rice

The 1 Mb tiling array was used to represent the 10.0 Mb to 11.0 Mb region of japonica rice Chromosome 10. A tile path was designed with 36-mer probes at a step of 5 nt. All 388,994 probes were retained and synthesized into a single array. This array was hybridized to the cDNA target used for the full-genome tiling array as well as targets prepared from non-coding RNA sample I and II. Keywords: noncoding RNA Non-coding RNA species were isolated from total RNA as previously described (5). Briefly, total RNA isolated from japonica rice leaves using the Trizol method was loaded onto an Qiagen-tip and eluted with 0.6~1.0M NaCl gradient of QRW2 buffer (Qiagen) at 50 oC. Fractions corresponding to tRNAs (sample I) and small RNAs of 80-500 nucleotides (nt) (sample II) were collected. To remove remaining mRNAs and rRNAs, the small RNA fractions were hybridized to a mixture of specifically designed oligos in the binding buffer. Unwanted RNA molecules were targeted and removed by a magnetic bead based process (MicrobExpress kit, Ambion). The enriched non-coding RNA pool was then dephosphorylated with a calf intestine alkaline phosphatase and ligated to a 3' adaptor oligonucleotide by the T4 RNA ligase (5). Small size molecules and excessive adaptors were removed from the ligation products using the mirVana miRNA isolation kit (Ambion) and reverse transcribed at 50 oC using the Thermoscript (Invitrogen) and the oligo 3RT as the primer (5). Amino-allyl-modified dCTP was incorporated into the cDNA strand during reverse transcription.