Multiplexed single-cell in situ RNA analysis by reiterative hybridization

Lu Xiao, Jia Guo

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)7290-7295
Number of pages6
JournalAnalytical Methods
Issue number17
StatePublished - Sep 7 2015

ASJC Scopus subject areas

  • Analytical Chemistry
  • Chemical Engineering(all)
  • Engineering(all)


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