A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection

Abeer K. Shaalan; Georgina M. Ellison-Hughes

doi:10.1016/j.xpro.2022.101231

A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection

Abeer K. Shaalan, Georgina M. Ellison-Hughes^*

^*Corresponding author for this work

GSTT Guy's and St Thomas' NHS Foundation Trust

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

Abstract

We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

Original language	English
Article number	101231
Journal	STAR Protocols
Volume	3
Issue number	1
Early online date	8 Mar 2022
DOIs	https://doi.org/10.1016/j.xpro.2022.101231
Publication status	Published - 18 Mar 2022

Keywords

Cell isolation
Gene Expression
Microscopy
Molecular Biology

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10.1016/j.xpro.2022.101231

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title = "A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection",

abstract = "We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.",

keywords = "Cell isolation, Gene Expression, Microscopy, Molecular Biology",

author = "Shaalan, {Abeer K.} and Ellison-Hughes, {Georgina M.}",

note = "Funding Information: This work was supported by a King's Together Fund Strategic Award from King's College London to G.M.E.-H. The authors would like to thank Prof. Abigail Tucker, Dean for Research and Professor of Development & Evolution, Centre for Craniofacial and Regenerative Biology, King's College London, for providing the mice used in this study. Publisher Copyright: {\textcopyright} 2022 The Author(s)",

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doi = "10.1016/j.xpro.2022.101231",

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N2 - We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

AB - We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

KW - Cell isolation

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KW - Microscopy

KW - Molecular Biology

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DO - 10.1016/j.xpro.2022.101231

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A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection

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Keywords

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