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.
Highlights
Multicellularity has evolved repeatedly across the tree of life and is a defining feature of land plants
In some plant species with C4 leaf anatomy, bundle sheath strands can be separated from the adjoining mesophyll by differential grinding (Covshoff, Furbank, Leegood, & Hibberd, 2013; Edwards & Black, 1971; Kanai & Edwards, 1973; Sheen, 1995)
Laser cap‐ ture microdissection was performed on an Arcturus Laser Capture Microdissection system using CapSure Macro Caps to collect bundle sheath strands and mesophyll cells
Summary
Multicellularity has evolved repeatedly across the tree of life and is a defining feature of land plants. Chemical fixation followed by paraffin embedding is the most com‐ monly used approach for laser capture microdissection of plant tis‐ sue and so has been used to study cell types from leaves of rice and maize, as well as tomato fruit, soybean roots and Arabidopsis flowers (Aubry, Kelly, et al, 2014; Aubry, Smith‐Unna, et al, 2014; Aubry et al, 2016; Gandotra, Coughlan, & Nelson, 2013; Jiao et al, 2009; Kerk et al, 2003; Klink, Alkharouf, MacDonald, & Matthews, 2005; Wuest & Grossniklaus, 2014) In these studies, non‐cross‐linking solutions such as Farmer's fixative or acetone are used to stabilize RNA, and the dehydrated tissue is mounted in paraffin wax at ~60°C to allow thin sections to be subjected to microdissection. By adopting a wax with a low‐melting‐point, as well as modifying sample preparation prior to microdissection and isolation of paradermal rather than transverse sections, we pro‐ vide a simple and robust method to allow high‐quality RNA to be obtained from specific cells of leaves that are not accessible using existing methodologies
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