Microarray-based analysis of microbial community RNAs by whole-community RNA amplification - PubMed (original) (raw)
Microarray-based analysis of microbial community RNAs by whole-community RNA amplification
Haichun Gao et al. Appl Environ Microbiol. 2007 Jan.
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 Deltafur 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.
Figures
FIG. 1.
Outline of the WCRA method. Gray type, RNA; black type, DNA. The primers used and other details are described in the text.
FIG. 2.
Evaluation of amplification biases using whole-genome S. oneidensis cDNA microarrays. (A) Primers tested in this study. N indicates a random nucleotide. (B) Ratios (amplified/unamplified) were plotted against the order of the genes in the genome, from SO0001 to SOA0173. In the unamplified panel, hybridization was carried out with the same unamplified RNA labeled with Cy3 and Cy5. In all other panels, 500 ng of RNA was amplified using WCRA with the primers indicated, labeled with Cy5, and hybridized with unamplified RNA (Cy3).
FIG. 3.
Scatter plots of genes from replicate hybridizations and replicate amplification reactions. (A to C) Scatter plots comparing the expression profiles of unamplified RNA from the wild-type strain (wt) with the expression profiles of unamplified RNA (A), 100 ng amplified starting RNA (B), or 50 ng amplified starting RNA (C) from the Δ_fur_ strain. (D) Quantitative analysis of relationships for expression ratios of all genes (Δ_fur_ RNA/wild-type RNA) between the amplified RNA (100 ng) and the unamplified RNA.
FIG. 4.
Amplification bias analysis by expression ratio comparison. All 30 genes examined have been reported to be highly affected by the fur mutation (36). (A) Expression ratios (Δ_fur_ RNA/wild-type RNA) for the genes obtained with unamplified RNA and 50 and 100 ng of the starting RNA. (B) Quantitative analysis of relationships for expression ratios of the 30 genes (Δ_fur_ RNA/wild-type RNA) between the amplified RNA and the unamplified RNA.
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