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Stress response study in Arabidopsis thaliana using DNA millichips

ABSTRACT: Our goal was to create a DNA chip that is as easy, convenient and inexpensive as an agarose gel. For a first generation solution, we describe a low cost, easy to use de novo synthesis oligonucleotide microarray technology that draws on the inherent flexibility of the maskless array synthesizer for in situ synthesis of thousands of photolithographically produced oligonucleotides covalently attached to a microscope slide. The method involves physically subdividing the slide into 1 x 1 mm 'millichips' that are hybridized to fluorescent RNA or DNA of biological origin, in a microfuge tube at an ordinary laboratory benchtop, rather than in dedicated hybridization chambers. Fluorescence intensity is then measured with a standard microxcope rather than sophisticated DNA chip scanners. For proof of principle, we measured changes in the transcriptome of Arabidopsis plants induced by growth in the presence of three major environmental abiotic stresses (temperature, light and water status), in all possible combinations. Validation by comparison with qRT-PCR showed a high correlation coefficient and analysis of variance indicated a high technical reproducibility. These experiments demonstrate that low cost DNA 'millichips' can be made and reliably used at the benchtop in a normal laboratory setting, without assistance of core facilities containing costly specialized instrumentation.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE37118 | GEO | 2012/04/12

SECONDARY ACCESSION(S): PRJNA158275

REPOSITORIES: GEO

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Stress response study in Arabidopsis thaliana using DNA millichips

Project description:Our goal was to create a DNA chip that is as easy, convenient and inexpensive as an agarose gel. For a first generation solution, we describe a low cost, easy to use de novo synthesis oligonucleotide microarray technology that draws on the inherent flexibility of the maskless array synthesizer for in situ synthesis of thousands of photolithographically produced oligonucleotides covalently attached to a microscope slide. The method involves physically subdividing the slide into 1 x 1 mm 'millichips' that are hybridized to fluorescent RNA or DNA of biological origin, in a microfuge tube at an ordinary laboratory benchtop, rather than in dedicated hybridization chambers. Fluorescence intensity is then measured with a standard microxcope rather than sophisticated DNA chip scanners. For proof of principle, we measured changes in the transcriptome of Arabidopsis plants induced by growth in the presence of three major environmental abiotic stresses (temperature, light and water status), in all possible combinations. Validation by comparison with qRT-PCR showed a high correlation coefficient and analysis of variance indicated a high technical reproducibility. These experiments demonstrate that low cost DNA 'millichips' can be made and reliably used at the benchtop in a normal laboratory setting, without assistance of core facilities containing costly specialized instrumentation. An average of three to four technical replicates and six biological replicates for each treatment and control group were performed resulting in a total of 457 individual millichip experiments for this study.

2012-04-11 | E-GEOD-37118 | biostudies-arrayexpress

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