SmiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability. Issue 22 (5th September 2016)

• Record Type: Journal Article
• Authors: Tsanov, Nikolay; Samacoits, Aubin; Chouaib, Racha; Traboulsi, Abdel-Meneem; Gostan, Thierry; Weber, Christian; Zimmer, Christophe; Zibara, Kazem; Walter, Thomas; Peter, Marion; Bertrand, Edouard; Mueller, Florian
• Is Part Of: Nucleic acids research. Volume 44:Issue 22(2016)
• Page Start: e165
• Page End: e165
• Publication Date: 2016-09-05
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkw784
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.