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- W3048314007 abstract "Single-cell RNA sequencing (scRNA-seq) and imaging technologies have revealed the transcriptomic diversity of single cells, the study of which is key to understanding biological processes and disease states. Alternative splicing of RNA contributes towards this diversity, yielding variable transcriptional profiles across different cell types within the human body. This diversity is challenging to visualize in situ because current isoform-sensitive RNA imaging methods are inefficient or are only applicable to genes with large isoform-specific exons. Here, we attempt to expand the toolbox of spatially informed RNA visualization methods by optimizing click-amplifying fluorescent in situ hybridization (clampFISH) to visualize alternatively-spliced isoforms and describe some of the challenges we encountered. We conducted a meta-analysis of combined short- and long-read sequencing data to select optimal targets for splicing analysis in human cell lines. We developed several single molecule FISH (smFISH) probe sets to evaluate efficacy of isoform-specific clampFISH probes in future experiments. Next, we modified clampFISH probes to include more landing pads (binding sites for subsequent probes) with the aim of achieving more rapid signal amplification with fewer primary probes. We observed higher target signal intensity with more landing pads, but this effect created higher off-target signal intensity as well. Finally, we tested whether adding magnesium ions to hybridization conditions would increase clampFISH probe specificity. The tested magnesium ion concentrations reduced nonspecific binding but also reduced specific binding. Our results lay the foundation for future experiments that will inform isoform distributions at a single-cell level.--Author's abstract" @default.
- W3048314007 created "2020-08-13" @default.
- W3048314007 creator A5078895108 @default.
- W3048314007 date "2021-05-10" @default.
- W3048314007 modified "2023-10-04" @default.
- W3048314007 title "Single-cell analysis of mRNA splicing variants" @default.
- W3048314007 doi "https://doi.org/10.17760/d20382869" @default.
- W3048314007 hasPublicationYear "2021" @default.
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