Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Non-coding RNA tiling array in rice

ABSTRACT: 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 3ï¢ 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.

ORGANISM(S): Oryza sativa

SUBMITTER: Xiangfeng Wang

PROVIDER: E-GEOD-6922 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Global identification and characterization of transcriptionally active regions in the rice genome.

Li Lei L Wang Xiangfeng X Sasidharan Rajkumar R Stolc Viktor V Deng Wei W He Hang H Korbel Jan J Chen Xuewei X Tongprasit Waraporn W Ronald Pamela P Chen Runsheng R Gerstein Mark M Deng Xing Wang XW

PloS one 20070314 3

Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approxim ...[more]

PMID: 17372628

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Project description:The number of annotated protein coding genes in the genome of Caenorhabditis elegans is similar to that of other animals, but the extent of its non-protein coding transcriptome remains unknown. Expression profiling on whole genome tiling microarrays applied to a mixed stage C. elegans population verified the expression of 71% of all annotated exons. Only a small fraction (11 %) of the polyadenylated transcription is non-annotated, and appears to consist of approximately 3200 missed or alternative exons and 7800 small transcripts of unknown function (TUFs). Almost half (44%) of the detected transcriptional output is non-polyadenylated and probably not protein coding, and of this 70% overlap the boundaries of protein coding genes in a complex manner. Specific analysis of small non-polyadenylated transcripts verified 98% of all annotated small ncRNAs, and suggested that the transcriptome contains about 1,200 small (<500 nt) unannoted non-coding loci. After combining overlapping transcripts, we estimate that at least 70% of the total C. elegans genome is transcribed. Keywords: RNA fractions RNA was extracted from mixed stage wild-type N2 strain worms cultivated at 20oC according to the Trizol (Invitrogen) protocol. Small RNAs (< 500nt, SNPA sample) were isolated using a Qiagen tip (Qiagen), and the Poly(A) Purist MAG (Ambion) and MicrobExpress kits (Ambion) were adapted to remove remaining mRNAs and rRNAs (Deng et al. 2006). The enriched ncRNA pool was cloned using an adaptor-mediated library construction protocol. RNAs were dephosphorylated with calf intestine alkaline phosphatase (Fermentas), then ligated to the 3M-bM-^@M-^Y-adaptor (3AD) oligonucleotide by T4 RNA ligase (Fermentas) (He et al. 2006). Polyadenylated RNA (PA sample) was isolated from total RNA using the Poly(A) Purist MAG kit (above). Non-polyadenylated RNA (NPA sample) was prepared by removing polyadenylated RNA using the Poly(A) Purist MAG kit and rRNA using the MicrobExpress kit.

Dataset Information

Non-coding RNA tiling array in rice

Publications

Global identification and characterization of transcriptionally active regions in the rice genome.

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