FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

Nature Protocols
Riccardo ArrigucciMaria Laura Gennaro

Abstract

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

References

May 9, 1998·Science·A M FeminoR H Singer
Aug 3, 2004·Nature Reviews. Immunology·Stephen P PerfettoMario Roederer
Mar 1, 2008·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Mario Roederer
Jul 25, 2008·Blood·Michael A Caligiuri
Sep 23, 2008·Nature Methods·Arjun RajSanjay Tyagi
Jul 4, 2009·Cytometry. Part B, Clinical Cytometry·Ruud HulspasD Robert Sutherland
Jun 26, 2010·Current Protocols in Cytometry·Stephen P PerfettoMario Roederer
Mar 25, 2011·Methods in Molecular Biology·Mona BatishSanjay Tyagi
Mar 7, 2012·Methods : a Companion to Methods in Enzymology·Pratip K Chattopadhyay, Mario Roederer
Nov 10, 2012·Nature Protocols·Stephen P PerfettoMario Roederer
Dec 5, 2012·PloS One·Hans M LarssonJeffrey A Hubbell
Aug 28, 2013·Methods in Molecular Biology·Samir Rahman, Daniel Zenklusen
Sep 12, 2013·Molecular Systems Biology·Khyati Shah, Sanjay Tyagi
Dec 7, 2013·Science·Alvaro Sanchez, Ido Golding
Dec 17, 2014·The Journal of Immunology : Official Journal of the American Association of Immunologists·Yuri BushkinSanjay Tyagi
Apr 30, 2015·Methods : a Companion to Methods in Enzymology·Steven McClellanLaurie Owen

❮ Previous
Next ❯

Citations

Sep 5, 2018·Lab on a Chip·Alexander M XuJames R Heath
Jun 13, 2019·PLoS Pathogens·Xiang YeJohn Karijolich
Oct 15, 2019·Immunological Reviews·Claire LoiseauDenise L Doolan
Jun 17, 2020·Nucleic Acids Research·Naoki UnoKatsunori Yanagihara
Mar 19, 2020·Chembiochem : a European Journal of Chemical Biology·Nada RaddaouiThomas Carell
Sep 19, 2019·Current Protocols in Neuroscience·Mona Batish, Sanjay Tyagi
Mar 29, 2020·PeerJ·Alexander P YoungRussell C Wyeth
Oct 5, 2018·Frontiers in Immunology·Riccardo ArrigucciMaria Laura Gennaro
Feb 26, 2020·Biosensors & Bioelectronics·Su WangZhiyang Li
May 1, 2021·International Journal of Molecular Sciences·Alessandra RossiSilvia Piconese
Jun 4, 2021·Communications Biology·Jayan RammohanDavid Ross
Jan 15, 2019·Analytical Chemistry·Meihua LinFan Xia

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.