Smifish and fish-quant – a flexible single rna detection approach with super-resolution capability
Tsanov, Nikolay Samacoits, Aubin Chouaib, Racha Traboulsi, Abdel-Meneem Gostan, Thierry Weber, Christian Zimmer, Christophe Zibara, Kazem Walter, Thomas Peter, Marion
Tsanov, Nikolay
Samacoits, Aubin
Chouaib, Racha
Traboulsi, Abdel-Meneem
Gostan, Thierry
Weber, Christian
Zimmer, Christophe
Zibara, Kazem
Walter, Thomas
Peter, Marion
Publication Date
2016-09-05
Type
Article
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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 (2016). Smifish and fish-quant – a flexible single rna detection approach with super-resolution capability. Nucleic Acids Research 44 (22),
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 fromprobe-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.
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Publisher
Oxford University Press (OUP)
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Attribution-NonCommercial-NoDerivs 3.0 Ireland