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An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material.

Published version
Peer-reviewed

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Authors

Hua, Lei 
Hibberd, Julian M 

Abstract

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low-melting-point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high-quality RNA suitable for down-stream applications such as quantitative reverse transcriptase-polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

Description

Keywords

RNA integrity and yield, bundle sheath strands, laser capture microdissection, mesophyll, rice leaves

Journal Title

Plant Direct

Conference Name

Journal ISSN

2475-4455
2475-4455

Volume Title

3

Publisher

Wiley
Sponsorship
Bill & Melinda Gates Foundation (via University of Oxford) (R38963/CN003)
The work was supported by a C4 Rice project grant from The Bill and Melinda Gates Foundation to the University of Oxford (2015-­2019).