Workshop Summary
シロイヌナズナとトウモロコシのRNA増幅および
マイクロアレイ解析

2003年3月からアリゾナ大学のマイクロアレイ施設でマイクロアレイワークショップが行なわれています(www.ag.arizona.edu/microarray)。年に二回、6日間の会議があり、研究者向けにマイクロアレイ実験のあらゆる側面からの実習トレーニングが計画され ています。このトレーニングには全米から集まったマイクロアレイの専門家やバイオ分野の統計学者による講演も 含まれます。ワークショップの参加者は、オリゴヌクレオチドのマイクロアレイを用いて最適な結果を得るための基礎知識を習得します。

The December 2004 workshop included lectures and laboratory work covering the following topics:

1. Experimental design

2. Total RNA isolation (tissue: plant seedlings)

3. RNA amplification (Amino Ally MessageAmp™ II Kit)

4. aRNA labeling (Cy™ dyes) and purification

5. Labeled aRNA hybridization to long oligonucleotide microarrays (University of Arizona Arabidopsis and Maize genome microarrays)

6. Microarray scanning

7. Data extraction, processing, and interpretation

Microarray Training Project

2004 Arizona Microarray Workshop participants studied gene expression profiles in untreated control plants and experimentally cold-treated plants (Arabidopsis and maize seedlings). Total RNA from plant seedlings was amplified and labeled using Ambion's Amino Allyl MessageAmp™ II Kit, which is based on the Van Gelder/Eberwine method. Analysis of the in vitro transcribed amino allyl aRNA products indicated a 3000-fold amplification of the mRNA, with an average size ranging from 1.5–2.5 kb (Figure 1). aRNA (4 µg) was coupled with Cy3 or Cy5 dyes and hybridized to the University of Arizona Arabidopsis and Maize genome microarrays (Figure 2). Microarray data were normalized using mixed model analysis of variance (ANOVA), and a modified t-test was applied to test the significance of definitely expressed genes. The results indicated that several hundred genes, including cold-induced genes like COR47, COR15, KIN1, RD29A, and ERD10, were up-regulated several fold in cold-treated plants.

Figure 1. Agilent® Bioanalyzer Traces of Amplified RNA from Arabidopsis and Maize Seedlings. Arabidopsis and Maize total RNA (1 µg) were converted into double-stranded cDNA; the cDNA was used in single round amplification (14 h) using Ambion's Amino Allyl MessageAmp™ II aRNA Amplification Kit. The recommended procedure was followed for RNA amplification with slight modification to the ratio of UTP:aaUTP. In vitro transcription reactions employed a 70% amino allyl UTP substitution for the in vitro transcription reaction. Heat denatured aRNA (100 ng) was analyzed using an Agilent RNA 6000 Nano LabChip® Kit: (A) Arabidopsis thaliana aRNA, and (B) Maize aRNA.

Figure 2. Images from the Arabidopsis Long Oligonucleotide Microarrays. Cy™3-labeled control and Cy5-labeled cold-treated Arabidopsis aRNA were hybridized (12 h at 55°C) to an Arabidopsis long oligonucleotide microarray.

Arizona Microarray Workshop

The exchange of ideas and techniques at these workshops is important for the growing microarray community to maximize accurate data collection and analysis. 38 researchers from Canada, China, Chile, Denmark, India, The Netherlands, South Africa, UK, and the USA participated in the December 2004 workshop. The University of Arizona Microarray Facility is committed to educating scientists and to providing cost-effective arrays for academic and non-profit researchers studying Arabidopsis and Maize expression profiling. The Arizona Microarray Workshop is funded in part by the National Science Foundation and has twelve industrial sponsors, including Ambion, Inc.

The 2005 Arizona Microarray Workshops
(May 8-13 and Fall—to be announced)

For more information about these biannual workshops or the data presented here, please visit

www.ag.arizona.edu/microarray/workshopMay2005.html

or contact Dr. David Galbraith (galbraith@arizona.edu)
or Dr. Rangasamy Elumalai (relu@ag.arizona.edu)
in the Department of Plant Sciences, University of Arizona.

Scientific Contributors
Rangasamy Elumalai • University of Arizona
Robert Setterquist • Ambion, Inc.

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