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Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays.

ABSTRACT: RNA catalytic and binding interactions with proteins and small molecules are fundamental elements of cellular life processes as well as the basis for RNA therapeutics and molecular engineering. In the absence of quantitative predictive capacity for such bioaffinity interactions, high throughput experimental approaches are needed to sufficiently sample RNA sequence space. Here we report on a simple and highly accessible approach to convert commercially available customized DNA microarrays of any complexity and density to RNA microarrays via a T7 RNA polymerase-mediated extension of photocrosslinked methyl RNA primers and subsequent degradation of the DNA templates.

SUBMITTER: Schaudy E

PROVIDER: S-EPMC9246885 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays.

Schaudy Erika E Hölz Kathrin K Lietard Jory J Somoza Mark M MM

Nature communications 20220630 1

RNA catalytic and binding interactions with proteins and small molecules are fundamental elements of cellular life processes as well as the basis for RNA therapeutics and molecular engineering. In the absence of quantitative predictive capacity for such bioaffinity interactions, high throughput experimental approaches are needed to sufficiently sample RNA sequence space. Here we report on a simple and highly accessible approach to convert commercially available customized DNA microarrays of any ...[more]

PMID: 35773271

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Project description:BackgroundRNA-Seq exploits the rapid generation of gigabases of sequence data by Massively Parallel Nucleotide Sequencing, allowing for the mapping and digital quantification of whole transcriptomes. Whilst previous comparisons between RNA-Seq and microarrays have been performed at the level of gene expression, in this study we adopt a more fine-grained approach. Using RNA samples from a normal human breast epithelial cell line (MCF-10a) and a breast cancer cell line (MCF-7), we present a comprehensive comparison between RNA-Seq data generated on the Applied Biosystems SOLiD platform and data from Affymetrix Exon 1.0ST arrays. The use of Exon arrays makes it possible to assess the performance of RNA-Seq in two key areas: detection of expression at the granularity of individual exons, and discovery of transcription outside annotated loci.ResultsWe found a high degree of correspondence between the two platforms in terms of exon-level fold changes and detection. For example, over 80% of exons detected as expressed in RNA-Seq were also detected on the Exon array, and 91% of exons flagged as changing from Absent to Present on at least one platform had fold-changes in the same direction. The greatest detection correspondence was seen when the read count threshold at which to flag exons Absent in the SOLiD data was set to t<1 suggesting that the background error rate is extremely low in RNA-Seq. We also found RNA-Seq more sensitive to detecting differentially expressed exons than the Exon array, reflecting the wider dynamic range achievable on the SOLiD platform. In addition, we find significant evidence of novel protein coding regions outside known exons, 93% of which map to Exon array probesets, and are able to infer the presence of thousands of novel transcripts through the detection of previously unreported exon-exon junctions.ConclusionsBy focusing on exon-level expression, we present the most fine-grained comparison between RNA-Seq and microarrays to date. Overall, our study demonstrates that data from a SOLiD RNA-Seq experiment are sufficient to generate results comparable to those produced from Affymetrix Exon arrays, even using only a single replicate from each platform, and when presented with a large genome.

Dataset Information

Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays.

Publications

Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays.

Similar Datasets

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