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Issue 17, 2015
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Multiplexed single-cell in situ RNA analysis by reiterative hybridization

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Abstract

Most current approaches for quantification of RNA species in their natural spatial contexts in single cells are limited by a small number of parallel analyses. Here we report a strategy to dramatically increase the multiplexing capacity for RNA analysis in single cells in situ. In this method, transcripts are detected by fluorescence in situ hybridization (FISH). After imaging and data storage, the fluorescence signal is efficiently removed by photobleaching. This enables the reinitiation of FISH to detect other RNA species in the same cell. Through reiterative cycles of hybridization, imaging and photobleaching, the identities, positions and copy numbers of a large number of varied RNA species can be quantified in individual cells in situ. Using this approach, we analyzed seven different transcripts in single HeLa cells with five reiterative RNA FISH cycles. This approach has the potential to detect over 100 varied RNA species in single cells in situ, which will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.

Graphical abstract: Multiplexed single-cell in situ RNA analysis by reiterative hybridization

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Supplementary files

Article information


Submitted
25 Feb 2015
Accepted
28 Apr 2015
First published
29 Apr 2015

This article is Open Access

Anal. Methods, 2015,7, 7290-7295
Article type
Paper
Author version available

Multiplexed single-cell in situ RNA analysis by reiterative hybridization

L. Xiao and J. Guo, Anal. Methods, 2015, 7, 7290
DOI: 10.1039/C5AY00500K

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