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. Researchers at Tsinghua University have designed 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. The researchers 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. This strategy provides a single-cell analysis platform to investigate the mechanism of complex immune responses and may further guide immunotherapy.
Schematic Diagram of Multiplex Detection of mRNA Variants in Single Cells by SpliceRCA
(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.