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. 2018 May 30;4(6):680–687. doi: 10.1021/acscentsci.8b00081

Scheme 1. Schematic Diagram of Multiplex Detection of mRNA Variants in Single Cells by SpliceRCA.

Scheme 1

(A) Alternative splicing patterns of CD45 during T-cell activation. Isoforms (CD45RA, CD45RB, CD45R0) with decreasing exon inclusion were expressed upon T-cell activation. (B) The procedures of SpliceRCA for detecting splice variants in single cells. The splice-junction anchored padlock probe is composed of four modules: the recognition of exon junction sites (Rx, Ry), universal priming region (P), and tag motif (T) modules. The newly formed splice junction in the target splice isoform brings close proximity between Rx and Ry in the padlock probe for circularizing, following primer hybridized with the P, triggering in situ RCA. Upon tuning of the sequence of T corresponding to different fluorophores, the three RNA splicing variants can thus be simultaneously differentiated, and visualized with single-molecule resolution attributed to the in situ one-target-one-amplicon amplification method.