La Trobe

File(s) not publicly available

Laser-capture microdissection RNA-sequencing for spatial and temporal tissue-specific gene expression analysis in plants

journal contribution
posted on 12.01.2021, 05:45 by Lim Liew, Yan Wang, Marta Peirats-Llobet, Oliver Berkowitz, James Whelan, Mathew Lewsey
© 2020 JoVE Journal of Visualized Experiments. The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

History

Publication Date

01/01/2020

Journal

Journal of Visualized Experiments

Volume

162

Issue

162

Article Number

e61517

Pagination

14p. (p. 1-14)

Publisher

MYJoVE Corporation

ISSN

1940-087X

Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.

Usage metrics

Journal Articles

Categories

Exports