title: Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232-8240, USA. joseph.watson@vanderbilt.edu
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Watson
authors.forename: Shenglong
authors.initials: S
authors.lastname: Wang
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Von Stetina
authors.forename: W Clay
authors.initials: WC
authors.lastname: Spencer
authors.forename: Shawn
authors.initials: S
authors.lastname: Levy
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Dexheimer
authors.forename: Nurith
authors.initials: N
authors.lastname: Kurn
authors.forename: Joe Don
authors.initials: JD
authors.lastname: Heath
authors.forename: David M
authors.initials: DM
authors.lastname: Miller
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D007814:Larva / Q000378:metabolism; D009420:Nervous System / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D018075:RNA, Complementary / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D018075:RNA, Complementary