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SpliceRCA: in Situ Single-Cell Analysis of mRNA Splicing Variants.
- Source :
-
ACS central science [ACS Cent Sci] 2018 Jun 27; Vol. 4 (6), pp. 680-687. Date of Electronic Publication: 2018 May 30. - Publication Year :
- 2018
-
Abstract
- Immune cell heterogeneity due to the differential expression of RNA splicing variants still remains unexplored. This is mainly because single-cell imaging technologies of splicing variants with precise sequence or base resolution are now not readily available. Herein, we design a splice-junction anchored padlock-probe-mediated rolling circle amplification assay (SpliceRCA) for single-cell imaging of splice isoforms of essential regulatory immune gene (CD45) upon T-cell activation. Two recognition regions in the padlock probe can target the splice-junction sequence, resulting in a close proximity for triggering in situ one-target-one-amplicon amplification. With the read length of ∼30 nucleotides, this method allows discrimination of isoforms with single-base precision and quantification of isoforms with single-molecule resolution. We applied SpliceRCA to single-cell image splice variants of essential regulatory immune gene (CD45) upon T-cell activation. It is found that CD45RO isoform presents a distal nuclear spatial distribution and is coregulated with CD45RB upon activation. Our strategy provides a single-cell analysis platform to investigate the mechanism of complex immune responses and may further guide immunotherapy.<br />Competing Interests: The authors declare no competing financial interest.
Details
- Language :
- English
- ISSN :
- 2374-7943
- Volume :
- 4
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- ACS central science
- Publication Type :
- Academic Journal
- Accession number :
- 29974063
- Full Text :
- https://doi.org/10.1021/acscentsci.8b00081