RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants
RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants
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RNA 引物扩增用于单细胞剪接变体的噪声抑制可视化
DOI：
10.1021/acs.analchem.0c01734
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发表时间：
2020
影响因子：
7.4
通讯作者：
Chen Feng
中科院分区：
文献类型：
--
作者：
Cao Xiaowen;Yu Huahang;Xue Jing;Bai Min;Zhao Yue;Li Youjun;Zhao Yongxi;Chen Feng
剪接变体可视化对于更深入地了解细胞生长和发育至关重要。然而，由于长度短、序列相似和丰度低，它在技术上仍然具有挑战性。现有的单细胞成像策略受到非特异性放大的影响，在剪接变体的可视化过程中会产生相当大的噪音。在此，我们开发了一种新的 RNA 引发的扩增策略，用于单细胞剪接变体的噪声抑制可视化。块探针设计用于特异性识别 mRNA 中外显子的缀合区域，然后用核酸内切酶消化该区域，并在 3' 末端提供羟基。 RNA靶标可以作为引物触发滚环扩增，实现剪接变体的可视化，并将噪声抑制到接近零。我们进一步探索了三种乳腺细胞系中 BRCA1 剪接变体的表达和分布，揭示了该抑癌基因的细胞类型特异性图谱。
Splice variants visualization is pivotal for a deeper understanding of cell growth and development. However, it remains technically challenging due to short lengths, similar sequences, and low abundance. The existing single-cell imaging strategies suffer from nonspecific amplification that causes considerable noise during visualization of the splice variants. Herein we develop a new RNA-primed amplification strategy for noise-suppressed visualization of single-cell splice variants. Block probes were designed to specifically identify the conjugated region of exons in mRNA, which was then digested by endonuclease and provided a hydroxyl group at the 3′ terminal. The RNA target can act as primer to trigger rolling circle amplification, achieving visualization of splice variants with noise suppressed to nearly zero. We further explored the expression and distribution ofBRCA1splice variants in three breast cell lines, revealing cell-type specific mapping of this cancer suppressor gene.