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Title: Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)

Abstract

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 {Delta}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 groundwatermore » 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.« less

Authors:
 [1];  [2];  [3];  [4];  [2];  [4]
  1. University of Oklahoma
  2. ORNL
  3. Texas A&M University
  4. University of Oklahoma, Norman
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
931177
DOE Contract Number:  
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 73; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ABUNDANCE; AMPLIFICATION; BIOREACTORS; COMMUNITIES; DENITRIFICATION; DETECTION; EVALUATION; FUNCTIONALS; GENES; MIXTURES; NUCLEOTIDES; RNA; STRAINS

Citation Formats

Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, and Zhou, Jizhong. Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA). United States: N. p., 2007. Web. doi:10.1128/AEM.01771-06.
Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, & Zhou, Jizhong. Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA). United States. doi:10.1128/AEM.01771-06.
Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, and Zhou, Jizhong. Mon . "Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)". United States. doi:10.1128/AEM.01771-06.
@article{osti_931177,
title = {Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)},
author = {Gao, Haichun and Yang, Zamin Koo and Gentry, Terry and Wu, Liyou and Schadt, Christopher Warren and Zhou, Jizhong},
abstractNote = {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 {Delta}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.},
doi = {10.1128/AEM.01771-06},
journal = {Applied and Environmental Microbiology},
number = 2,
volume = 73,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}