Although it is now possible to visualize individual RNA molecules within cells by fluorescent in situ hybridization, the resulting signals are quite weak. Here we describe a method that produces amplified signals from individual RNA molecules, while minimizing false positive signals.
Our probes allow the detection of poorly expressed mRNAs, and are so specific that related wild-type and mutant mRNAs, differing from each other by only a single nucleotide variation, produce distinct in situ signals in 2 distinguishable colors.
This technology will help studies of gene expression phenomena, such as allelic imbalance, and will be useful for the identification of mRNAs that harbor somatic mutations in cancer cells, facilitating targeted therapy.
Abstract
Amplification of signals by hybridization chain reaction (HCR) is a powerful approach to increase signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR primer sequence are prone to produce false signals.
Here we describe an interacting binary hairpin probe system in which the HCR primer sequence is conditionally sequestered. Binding of these probes to a perfectly complementary target unmasks the primer, allowing the generation of an amplified signal. This probe system can distinguish single nucleotide variations within individual mRNA molecules and produces amplified signals in situ for wild-type and mutant variants, each in a distinguishable color.
This technology will increase studies of unbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By placing these probes along an mRNA target, enhanced signals can be obtained, allowing the scanning of tissue sections for gene expression using lower-power microscopy, overcoming tissue autofluorescence and allowing the detection of biomarkers of low abundance on flow cytometry.