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Applied and Environmental Microbiology, January 2007, p. 563-571, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01771-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Microarray-Based Analysis of Microbial Community RNAs by Whole-Community RNA Amplification

Haichun Gao,^1,2,3 Zamin K. Yang,¹ Terry J. Gentry,^1,4 Liyou Wu,^1,3 Christopher W. Schadt,¹ and Jizhong Zhou^1,3*

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee,¹ Center for Microbial Ecology, Michigan State University, East Lansing, Michigan,² Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019,³ Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas⁴

Received 26 July 2006/ Accepted 2 November 2006

A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis fur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.

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* Corresponding author. Mailing address: Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019. Phone: (405) 325-6073. Fax: (405) 325-3442. E-mail: jzhou{at}ou.edu.

Published ahead of print on 10 November 2006.

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Applied and Environmental Microbiology, January 2007, p. 563-571, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01771-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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