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Detection of SINEUP RNA modification by Oxford Nanopore direct-RNA sequencing

ABSTRACT: To detect the modifed bases in SINEUP RNA, we compared chemically modified in vitro transcribed (IVT) SINEUP-GFP RNA and in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells. Comparative study of Nanopore direct RNA sequencing data from non-modified and modified IVT samples against the data from ICT SINEUP RNA sample revealed modified k-mers positions in SINEUP RNA in the cell.

ORGANISM(S): Homo sapiens

PROVIDER: GSE224018 | GEO | 2023/02/04

REPOSITORIES: GEO

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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

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Detection of SINEUP RNA modification by Oxford Nanopore direct-RNA sequencing

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