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Open Access Methodology article

Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system

Joseph D Watson12, Shenglong Wang3, Stephen E Von Stetina1, W Clay Spencer1, Shawn Levy4, Phillip J Dexheimer4, Nurith Kurn3, Joe Don Heath3 and David M Miller12*

Author Affiliations

1 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232-8240, USA

2 Graduate Program in Neuroscience, Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37232-8548, USA

3 NuGEN Technologies, Inc., San Carlos, CA 94070, USA

4 Department of Biomedical Informatics, Vanderbilt University, Nashville, TN 37232-8240, USA

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BMC Genomics 2008, 9:84  doi:10.1186/1471-2164-9-84

Published: 19 February 2008

Abstract

Background

DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system.

Results

We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of pan-neural RNA. WT-Pico results in a higher fraction of present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification.

Conclusion

We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting.