Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos

This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of the brains of the fly Drosophila melanogaster and other insects, e.g. the jumping ant Harpegnathos saltator. Probes and HCR reagents are purchased from Molecular Instruments. This protocol is loosely based on the "generic sample in solution" protocol published by Molecular Instruments. Our modifications include the description of fixation conditions, counterstaining by Hoechst, and altered washes. Additionally, we use larger concentrations of probes and hairpins following the protocol described by Younger, Herre et al. 2020. We have successfully employed this protocol to stain insect brains with up to 4 different probe sets simultaneously (hairpins conjugated with Alexa Fluor 488, 546, 496, and 647).

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smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

10.1101/2020.02.29.971390 ◽

2020 ◽

Author(s):

Llilians Calvo ◽

Matthew Ronshaugen ◽

Tom Pettini

Keyword(s):

In Situ Hybridization ◽

Single Cell ◽

Single Molecule ◽

Fluorescent In Situ Hybridization ◽

Single Cells ◽

Fano Factor ◽

Confocal Imaging ◽

Cell Segmentation ◽

Expression Variability

ABSTRACTRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualisation and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

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Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization

Nature Protocols ◽

10.1038/nprot.2013.066 ◽

2013 ◽

Vol 8 (6) ◽

pp. 1100-1113 ◽

Cited By ~ 91

Author(s):

Samuel O Skinner ◽

Leonardo A Sepúlveda ◽

Heng Xu ◽

Ido Golding

Keyword(s):

Escherichia Coli ◽

In Situ Hybridization ◽

Single Molecule ◽

Copy Number ◽

Fluorescent In Situ Hybridization ◽

Mrna Copy Number ◽

Escherichia Coli Cells

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ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

Scientific Reports ◽

10.1038/s41598-020-76439-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Mengwei Hu ◽

Bing Yang ◽

Yubao Cheng ◽

Jonathan S. D. Radda ◽

Yanbo Chen ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Single Molecule ◽

Probe Design ◽

Powerful Method ◽

Rna Fish ◽

Technological Advances ◽

Multiplexed Imaging ◽

One Stop

AbstractFluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community.

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