Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

Lim Chee Liew; Yan Wang; Marta Peirats-Llobet; Oliver Berkowitz; James Whelan; Mathew  G. Lewsey

doi:10.3791/61517

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expres...

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Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

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08:33 min

August 5, 2020

DOI: 10.3791/61517-v

Lim Chee Liew^1,2, Yan Wang^1,2, Marta Peirats-Llobet¹, Oliver Berkowitz^1,2,3, James Whelan^1,2,3, Mathew G. Lewsey^1,3

¹Department of Animal, Plant and Soil Science, AgriBio Building,La Trobe University, ²Australian Research Council Centre of Excellence in Plant Energy Biology,La Trobe University, ³Australian Research Council Research Hub for Medicinal Agriculture, AgriBio Building,La Trobe University

Overview

This study presents a protocol for Laser-Capture Microdissection (LCM) coupled with RNA sequencing to analyze spatial and temporal transcriptomes in plant cells. The technique allows for precise isolation of cells within their native tissue context, facilitating detailed gene expression studies.

Key Study Components

Research Area

Plant biology
Molecular biology
Transcriptomics

Background

Importance of tissue-specific gene expression analysis
Need for contamination-free techniques in sample preparation
Benefits of RNA sequencing in understanding plant biology

Methods Used

Laser-Capture Microdissection (LCM)
Plant cells, specifically from barley seeds
RNA extraction and sequencing

Main Results

Successfully isolated specific cells for RNA analysis
Identified gene expression patterns over 48 hours during germination
Demonstrated the effectiveness of LCM for high-quality RNA samples

Conclusions

The protocol enhances understanding of spatial and temporal gene expression in plants.
It provides a valuable method for researchers studying developmental biology and genetics.

Frequently Asked Questions

What is Laser-Capture Microdissection?

Laser-Capture Microdissection is a technique used to isolate specific cells from tissue sections with minimal contamination.

How does RNA sequencing complement LCM?

RNA sequencing allows for the analysis of gene expression profiles from the isolated cells, providing insights into their function and behavior.

What types of samples can be processed with this protocol?

While optimized for plant cells, the protocol can be adapted for various tissue types that can be histologically identified.

Why is sample preparation critical in this process?

Proper fixation and embedding are essential to maintain tissue integrity and ensure successful dissection and RNA extraction.

What are the key findings of this research?

The research highlights distinct gene expression patterns in various plant tissues during germination, revealing insights into developmental processes.

How long does the protocol take?

The entire process, including sample preparation and RNA sequencing, requires careful management of time but typically spans several days.

What are the implications of this research?

The study contributes to our understanding of gene regulation in plants, which is crucial for fields like agriculture and biotechnology.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

This protocol uses Laser-Capture Microdissection coupled with RNA-Sequencing to obtain Spatial and Temporal transcriptomes from specific cells in plants of interests, using small quantities of biological materials. The main advantage of laser technique is that it facilitates the direct visualization of cells within normal tissue context, allowing the discrete cells to be precisely isolated in a contact free manner. Although this protocol is optimized for the isolation of plant cells, it can be applied to most cells that can be stologically identified.

Good sample preparation is critical. Therefore, one performing this technique for the first time, prop optimization of the tissue fixation and the embedding is important before Laser-Capture Microdissection. Before collecting the tissue sample, prepare a fixative appropriate to the species and tissue type to be harvested.

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Laser-Capture Microdissection RNA-Sequencing Spatial Transcriptomes Temporal Transcriptomes Plant Gene Expression Sample Preparation Barley Seed Processing Ethanol Gradient Dehydration Xylene Immersion Paraffin Embedding Histological Identification