smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.
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IF: 19.160
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Cited by: 267
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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.

Keywords

Gene Expression
smFISH
smiFISH

MeSH terms

Animals
Gene Expression Profiling
HeLa Cells
Humans
In Situ Hybridization, Fluorescence
Limit of Detection
Mice
Microscopy, Fluorescence
Mouse Embryonic Stem Cells
RNA, Messenger

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

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