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Highly Specific Multiplexed RNA Imaging in Tissues with Split-FISH

We present split-FISH, a multiplexed fluorescence in situ hybridization method that leverages a split-probe design to achieve enhanced specificity. Split-FISH reduces off-target background fluorescence, decreases false positives and enables accurate RNA profiling in uncleared tissues. We demonstrate the efficacy of split-FISH on various mouse tissues by quantifying the distribution and abundance of 317 genes in single cells and reveal diverse localization patterns for spatial regulation of the transcriptome in complex tissues.
Learning Objectives

  • Multiplexed FISH is an attractive tool for spatial transcriptomics.
  • Advantages of split-FISH design in lowering background and false positive noise
  • Example applications in profiling RNA localizations in mouse organs

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