Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptional Analysis of 1M Regions in Medicago truncatula Using Tiling Microarrays

ABSTRACT: We report here a transcriptonal analysis in six different organ types of a approximately 1 Mb region in legume plants barrel medic (Medicago truncatula) which is sytenic with soybean (Glycine max). We used oligonucleotide tiling microarrays to detecte transcription of over 80% of the predicted genes in both species. We detected differential gene expression in the six examined organ types. Keywords: RNA Both the barrel medic and soybean tiling arrays were produced on the Maskless Array Synthesizer platform. Briefly, tiling-paths consisting of 36-mer oligonucleotides offset by five nucleotides were designed to represent both DNA strands of the selected barrel medic and soybean genome sequence. Probes were synthesized at a feature-density of 390,000 probes per array in a âchess boardâ design. Microarray production and storage were carried out. Total RNA and mRNA were sequentially isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen) according to the manufacturerâs recommendations, respectively. mRNA from different organ types was reverse transcribed using a mixture of oligo(dT)18 and random nonamer primers, during which amino-allyl-modified dUTP (aa-dUTP) was incoporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare, Piscataway, NJ) to the amino-allyl functional groups in the cDNA. Two Î¼g dye-labeled targets were used for hybridization.

ORGANISM(S): Medicago truncatula

SUBMITTER: XingWang Deng

PROVIDER: E-GEOD-10055 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays.

Li Lei L He Hang H Zhang Juan J Wang Xiangfeng X Bai Sulan S Stolc Viktor V Tongprasit Waraporn W Young Nevin D ND Yu Oliver O Deng Xing-Wang XW

Genome biology 20080319 3

<h4>Background</h4>Legumes are the third largest family of flowering plants and are unique among crop species in their ability to fix atmospheric nitrogen. As a result of recent genome sequencing efforts, legumes are now one of a few plant families with extensive genomic and transcriptomic data available in multiple species. The unprecedented complexity and impending completeness of these data create opportunities for new approaches to discovery.<h4>Results</h4>We report here a transcriptional a ...[more]

PMID: 18348734

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Project description:In the study of gene expression regulation in plants, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array includes all pairs of 25-mer allele-specific probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Hybridization of the tiling array to a titration series in which the japonica- and indica-derived cDNA templates are mixed with altering proportions and development of a novel bioinformatic methodology allowed us to screen for diagnostic probe pairs for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica × japonica F1 hybrids and detected imbalanced expression at 95 (33%) and 71 (25%) SNPs, respectively. Comparison results from RNA-sequencing and allele-specific real-time PCR experiment validates the sensitivity and reliability of the tiling array method. Together, our results demonstrate the advantages of the tiling array method in interrogating large numbers of SNPs and for the reliability of the experimental and analytical techniques used for quantifying allele-specific gene expression. For each selected SNP, 25 pairs of 25-mer oligonucleotide probes were designed with one match perfectly to the indica allele and the other the japonica allele within each pair. Among the 25 probe pairs, the polymorphic site was placed at a different position such that all possible 25-mer allele-specific probes spanning the polymorphic site were included. The resultant 25,900 probes were synthesized in triplicate in a single microarray produced on the Maskless Array Synthesizer platform as previously described (Li et al., 2005; 2006). Poly(A+) RNA from japonica, indica and their reciprocal hybrids was reverse transcribed using an oligo(dT)18 primer, during which amino-allyl-modified dUTP (aa-dUTP) was incorporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare) to the amino-allyl functional groups in the cDNA. Dye-labeled target was quantified using a spectrophotometer and two μg used for hybridization to each array. In the titration series experiment, the dye-labeled targets from japonica and indica were mixed in known proportions before hybridization.

Dataset Information

Transcriptional Analysis of 1M Regions in Medicago truncatula Using Tiling Microarrays

Publications

Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays.

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