Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of C.elegans larval nervous system amplified by two different methods

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. Experiment 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.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: David Miller

PROVIDER: E-GEOD-9485 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project 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

Project description:Background: With its fully sequenced genome and simple, well-defined nervous system, the nematode C. elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and thereby link comprehensive profiles of neuronal transcription to key developmental and functional attributes of the nervous system. Results: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Micro-Array Profiling C. elegans Cells) method, we used Fluorescence Activated Cell Sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (e.g. transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. In addition to utilizing these profiling approaches to define stage specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. Conclusion: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and therefore provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system. Experiment Overall Design: Our goal is to profile gene expression throughout the nervous system of the model organism Caenorhabditis elegans. As a first goal, we profiled the entire nervous system at two developmental timepoints. To isolate transcripts from embryonic neurons we employed the MAPCeL (Microarray Profiling C. elegans Cells) technique in which pan-neuronal GFP+ cells are captured by FACS for RNA isolation. Since postembryonic cells are unattainable via this method, we used mRNA-tagging to isolated 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. Following formaldehyde cross-linking, animals were passed through a French press and Dounce homogenized to isolate a cell-free extract. Anti-FLAG antibodies were incubated with the cell-free extract to capture FLAG-PAB bound RNAs. After elution and reversal of the crosslinks, RNAs are isolated by TriZOL extraction. We also identified transcripts enriched in a subset of C. elegans larval neurons, the A-class neurons (using unc-4::3xFLAG::PAB-1). Experiment Overall Design: 3 sets of experiments normalized separately by RMA Experiment Overall Design: larval references of sets 2 and 3 originate from the same hybridizations (accompanying CEL files are identical)

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Transcription profiling of C.elegans larval nervous system amplified by two different methods

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