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Title: cRNA amplification methods enhance microarray identification of transcripts expressed in the nervous system      
keywords:
Transcriptome or Gene expression
ID:
PRJNA103265
description:
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. Keywords: expression profile Overall design: We profiled gene expression throughout the nervous system of the model organism Caenorhabditis elegans using two different amplification methods, WT-Pico and IVT (in vitro transcription). Since postembryonic cells are unattainable via this method, we used mRNA-tagging to isolate pan-neural RNAs from larval animals. In short, an epitope tagged poly-A binding protein (FLAG-PAB-1) was driven in all neurons using the F25B3.3 promoter. We then amplified harvested RNA using IVT or WT-Pico. Data generated from this study reveals significant differences between the two amplification methods. However, both seem to identify bona fide neural transcripts. Our work suggests that using two amplification methods, when possible, will provide one with a more accurate view of transcription in a particular cell type.
accesstypes:
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA103265
authentication:
none
authorization:
none
ID:
pmid:18284693
dateReleased:
11-01-2007
name:
Caenorhabditis elegans
ncbiID:
ncbitax:6239
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject
  • T32 MH064913/MH/NIMH NIH HHS/United States

  • P01 DK58212/DK/NIDDK NIH HHS/United States

  • P60 DK020593/DK/NIDDK NIH HHS/United States

  • P30 EY08126/EY/NEI NIH HHS/United States

  • F31 NS049743/NS/NINDS NIH HHS/United States

  • R01 NS026115/NS/NINDS NIH HHS/United States

  • P30 CA68485/CA/NCI NIH HHS/United States

  • U01 HG004263/HG/NHGRI NIH HHS/United States

  • P30 DK058404/DK/NIDDK NIH HHS/United States

  • P30 HD015052/HD/NICHD NIH HHS/United States

  • P01 HL6744/HL/NHLBI NIH HHS/United States

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