Unknown,Transcriptomics,Genomics,Proteomics

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Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression

ABSTRACT: 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.

ORGANISM(S): Oryza sativa

SUBMITTER: Hang He

PROVIDER: E-GEOD-20678 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression.

He Hang H Zhang Huiyong H Wang Xiangfeng X Wu Nicholas N Yang Xiaozeng X Chen Runsheng R Li Yi Y Deng Xing Wang XW Li Lei L

Genomics 20100803 5

In the study of gene expression, 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 included all 25-mer probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. T ...[more]

PMID: 20688151

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Project description:Rice in tropical and sub-tropical areas is often subjected to cold stress at the seedling stage resulting in poor growth and yield loss. In general, japonica rice has stronger cold tolerance (CT) than indica rice. However, several favorite alleles for CT exist in indica rice and can be used to enhance CT under japonica background. Genome-wide gene expression profiling is the efficient way to decipher the molecular genetic mechanism of CT enhancement and provide valuable information for the CT improvement in rice molecular breeding. In this study, the transcriptome of a CT introgressed line (IL) K354 and its recurrent parent C418 under cold stress were comparatively analyzed to explore the possible CT enhancement mechanism of K354.Totally 3184 differentially expressed genes (DEGs) including 195 transcription factors were identified in both lines under cold stress, about half of them were commonly regulated, which were involved in the major cold responsive pathways including OsDREB1s regulon. The K354-specific cold-induced DEGs were mainly related to stimulus response, cellular cell wall organization, and microtubule-based movement process compared with commonly cold-induced DEGs by GO analysis. Moreover, 296 constitutive DEGs with significantly different transcription level between C418 and K354 were detected under both control and cold stress conditions. K354-specific cold-regulated and constitutive DEGs jointly account for the CT improvement of K354. Pathway analysis unraveled up-regulation of starch and sucrose metabolism in both genotypes and presumably weaker defense response to stress in K354 than C418 under cold stress. Candidate genes prediction based on previous putative CT genetic networks revealed genotype-dependent CT enhancement mechanism in CT IL K354 vs recurrent parent C418, including Sir2, OsFAD7, OsWAK112d, and PCD related genes, etc.We propose a hypothesis of the CT enhancement mechanism in rice based on the results in present study. Firstly, a number of cold-regulated genes are able to express constitutively at high level or absent under control condition for standing by the arrival of cold stress. Next, under cold stress slower perception of stress signal from the more cold-tolerant membrane can postpone the activation of defensive system which may have possible negative effects on rice growth. Then, PCD will be launched to sacrifice a few cells for maintaining the basal growth of most cells. Finally, the protective responses on multiple aspects of cold damage will be postponed because of delayed several cold-defensive pathways (i.e. OsDREB1C regulon). It might explain why the recovery capacity of K354 from cold stress to control condition is stronger than C418. The CT enhancement mechanism can be regarded as the possible way to improve CT of japonica rice using indica germplasm in rice breeding program. In this study, the specific gene expression patterns of two genotypes (C418 and K354) at 2h, 6h, 12h, 24h and 48h during cold stress treatment (4 C) and control were characterized by using the Affymetrix rice microarray platform.

Dataset Information

Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression

Publications

Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression.

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