A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis

Stian Olsen,Kirsten Krause

doi:10.1186/s13007-019-0471-3

Abstract

BackgroundGene expression changes that govern essential biological processes can occur at the cell-specific level. To gain insight into such events, laser microdissection is applied to cut out specific cells or tissues from which RNA for gene expression analysis is isolated. However, the preparation of plant tissue sections for laser microdissection and subsequent RNA isolation usually involves fixation and embedding, processes that are often time-consuming and can lower the yield and quality of isolated RNA.ResultsInfection sites of the parasitic plant Cuscuta reflexa growing on its compatible host plant Pelargonium zonale were sectioned using a vibratome and dried on glass slides at 4 °C before laser microdissection. High quality RNA (RQI > 7) was isolated from 1 mm2, 3 mm2 and 6 mm2 total surface areas of laser microdissection-harvested C. reflexa tissue, with the yield of RNA correlating to the amount of collected material (on average 7 ng total RNA/mm2). The expression levels of two parasite genes previously found to be highly expressed during host plant infection were shown to differ individually between specific regions of the infection site. By drying plant sections under low pressure to reduce the dehydration time, the induced expression of two wound-related genes during preparation was avoided.ConclusionsPlants can be prepared quickly and easily for laser microdissection by direct sectioning of fresh tissue followed by dehydration on glass slides. We show that RNA isolated from material treated in this manner maintains high quality and enables the investigation of differential gene expression at a high morphological resolution.

Highlights

Gene expression changes that govern essential biological processes can occur at the cell-specific level
Plant organs typically consist of multiple cell and tissue types, and there is an increasing awareness that essential gene expression changes occurring in specific cells or tissues can be overlooked when RNA is isolated from entire organs [1,2,3]
With regards to performing gene expression analysis on RNA isolated from Laser microdissection (LM)-collected material, both the quantity and the quality of the isolated RNA are of importance

Summary

Introduction

Gene expression changes that govern essential biological processes can occur at the cell-specific level. To gain insight into such events, laser microdissection is applied to cut out specific cells or tissues from which RNA for gene expression analysis is isolated. The preparation of plant tissue sections for laser microdissection and subsequent RNA isolation usually involves fixation and embedding, processes that are often time-consuming and can lower the yield and quality of isolated RNA. Plant organs typically consist of multiple cell and tissue types, and there is an increasing awareness that essential gene expression changes occurring in specific cells or tissues can be overlooked when RNA is isolated from entire organs [1,2,3]. Gotte et al [15] demonstrated that gene expression analysis can be performed on LM-harvested plant material without prior fixation, albeit tissues were not sectioned but directly laser microdissected

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Discussion

Conclusion