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Nucleic Acids Res. 2016 Dec 15;44(22):e165. Epub 2016 Sep 5.

smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

Author information

1
Institut de Génétique Moléculaire de Montpellier, UMR 5535 CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France.
2
Unité Imagerie et Modélisation, Institut Pasteur and CNRS UMR 3691, 28 rue du Docteur Roux, 75015 Paris, France.
3
C3BI, USR 3756 IP CNRS - Paris, France.
4
Université de Montpellier, 163 rue Auguste Broussonnet, 34090 Montpellier, France.
5
ER045, Laboratory of Stem Cells, DSST, PRASE, Lebanese University, Beirut, Lebanon.
6
Biology Department, Faculty of Sciences-I, Lebanese University, Beirut, Lebanon.
7
MINES ParisTech, PSL-Research University, CBIO-Centre for Computational Biology, 77300 Fontainebleau, France.
8
Institut Curie, 75248 Paris Cedex, France.
9
INSERM, U900, 75248 Paris Cedex, France.
10
Institut de Génétique Moléculaire de Montpellier, UMR 5535 CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France marion.peter@igmm.cnrs.fr.
11
Institut de Génétique Moléculaire de Montpellier, UMR 5535 CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France edouard.bertrand@igmm.cnrs.fr.
12
Unité Imagerie et Modélisation, Institut Pasteur and CNRS UMR 3691, 28 rue du Docteur Roux, 75015 Paris, France fmueller@pasteur.fr.

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.

PMID:
27599845
PMCID:
PMC5159540
DOI:
10.1093/nar/gkw784
[Indexed for MEDLINE]
Free PMC Article

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