SpliceRCA: in Situ Single-Cell Analysis of mRNA Splicing Variants,ACS Central Science

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SpliceRCA: in Situ Single-Cell Analysis of mRNA Splicing Variants
ACS Central Science ( IF 12.7 ) Pub Date : 2018-05-30 00:00:00 , DOI: 10.1021/acscentsci.8b00081
Xiaojun Ren _{1,

2} , Ruijie Deng ₁ , Kaixiang Zhang ₁ , Yupeng Sun ₁ , Xucong Teng ₁ , Jinghong Li ₁

Affiliation

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.

中文翻译：

SpliceRCA：mRNA剪接变体的原位单细胞分析

由于RNA剪接变体的差异表达而导致的免疫细胞异质性仍未探索。这主要是因为现在尚不容易获得具有精确序列或碱基分辨率的剪接变体的单细胞成像技术。在本文中，我们设计了一个剪接点锚定的挂锁探针介导的滚环扩增测定法（SpliceRCA），用于在T细胞活化后对基本调节免疫基因（CD45）的剪接亚型进行单细胞成像。挂锁探针中的两个识别区域可以靶向剪接点序列，从而非常接近就地触发一靶一扩增子扩增。该方法具有约30个核苷酸的读取长度，可通过单碱基精度区分同工型，并以单分子分辨率对同工型进行定量。我们将SpliceRCA应用于T细胞活化后必需调节免疫基因（CD45）的单细胞图像拼接变体。发现CD45RO同工型呈现远端核空间分布，并在活化时与CD45RB共粒化。我们的策略提供了一个单细胞分析平台来研究复杂的免疫反应机制，并可能进一步指导免疫治疗。

更新日期：2018-05-30

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