Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos

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

Scientific Reports ◽

10.1038/s41598-018-26345-0 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 8

Author(s):

Fang Xie ◽

Kelsey A. Timme ◽

Jennifer R. Wood

Keyword(s):

In Situ Hybridization ◽

Single Molecule ◽

Fluorescent In Situ Hybridization ◽

Rna Fish

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

Communications Biology ◽

10.1038/s42003-021-01803-0 ◽

2021 ◽

Vol 4 (1) ◽

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 visualization 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.

Download Full-text