Imaging of individual transcripts by amplification-based single-molecule fluorescence in situ hybridization

•A novel method termed asmFISH for in situ imaging of individual RNA molecules has been developed.•AsmFISH provides improved detection efficiency and specificity.•AsmFISH can be used for in situ SNP genotyping.•AsmFISH is applicable to fixed cell samples as well as fresh frozen or FFPE tissue sections. An amplification-based single-molecule fluorescence in situ hybridization (asmFISH) assay is introduced that exploits improved probe design for highly specific imaging of individual transcripts in fixed cells and tissues. In this method, a pair of DNA ligation probes are ligated on RNA templates upon specific hybridization, followed by probe circularization based on enzymatic DNA ligation and rolling circle amplification for signal boosting. The method is more efficient and specific than the padlock probe assay for detection of the same RNA molecules and discrimination of single nucleotide polymorphisms. Moreover, asmFISH is a versatile method which can be applied not only to cultured cells, but also to fresh frozen and formalin-fixed, paraffin-embedded tissue sections.