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: 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.

Project description:Background RNA sequencing (RNA-seq) is a powerful technique for identifying and quantifying transcription and splicing events, both known and novel. However, given its recent development and the proliferation of library construction methods, understanding the bias it introduces is incomplete but critical to realizing its value. Results Here we present a method, in vitro transcription sequencing (IVT-seq), for identifying and assessing the technical biases in RNA-seq library generation and sequencing at scale. We created a pool of > 1000 in vitro transcribed (IVT) RNAs from a full-length human cDNA library and sequenced them with poly-A and total RNA-seq, the most common protocols. Because each cDNA is full length and we show IVT is incredibly processive, each base in each transcript should be equivalently represented. However, with common RNA-seq applications and platforms, we find ~50% of transcripts have > 2-fold and ~10% have > 10-fold differences in within-transcript sequence coverage. Strikingly, we also find > 6% of transcripts have regions of high, unpredictable sequencing coverage, where the same transcript varies dramatically in coverage between samples, confounding accurate determination of their expression. To get at causal factors, we used a combination of experimental and computational approaches to show that rRNA depletion is responsible for the most significant variability in coverage and that several sequence determinants also strongly influence representation. Conclusions In sum, these results show the utility of IVT-seq in promoting better understanding of bias introduced by RNA-seq and suggest caution in its interpretation. Furthermore, we find that rRNA-depletion is responsible for substantial, unappreciated biases in coverage. Perhaps most importantly, these coverage biases introduced during library preparation suggest exon level expression analysis may be inadvisable. 5 rRNA-depleted samples with duplicates, 1 polyA selected, 1 total RNA, and 1 plasmid library all without replicates.

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Variability in Microarray Labeling Methods

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