@inbook{12edb87dbb424187b2e152480bec9c33,
title = "Quantitative fluorescence in situ hybridization detection of plant mRNAs with single-molecule resolution",
abstract = "Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH spots that reflect the copy number of target RNA.",
keywords = "Autofluorescence, Confocal, FISH, In situ hybridization, Linear spectral unmixing, Plant, Quantification, RNAs, Single-molecule, Spectra",
author = "Kun Huang and Mona Batish and Chong Teng and Alex Harkess and Meyers, {Blake C.} and Caplan, {Jeffrey L.}",
note = "Funding Information: This project was supported by the US NSF Plant Genome Research Program, awards 1649424, 1611853, and 1754097. We would like to thank members of the Batish lab for input on single-molecule in situ hybridization, and members of the Meyers and Caplan labs for help and support. Microscopy equipment was acquired with a shared instrumentation grant (S10 OD016361) and access was supported by the NIH-NIGMS (P20 GM103446), the NSF (IIA-1301765), and the State of Delaware. Publisher Copyright: {\textcopyright} Springer Science+Business Media, LLC, part of Springer Nature 2020.",
year = "2020",
doi = "10.1007/978-1-0716-0712-1_2",
language = "English",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "23--33",
booktitle = "Methods in Molecular Biology",
}