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

Summary

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.

Abstract

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.

Introduction

Plant development and growth involve the coordinated action of transcriptional regulatory networks within different cells that exist in a complex cellular environment. To understand the activity of these regulatory networks, we require the knowledge of spatial and temporal gene expression within different cell types across developmental stages. However, analyses of gene expression are more commonly conducted in whole organs or bulk tissue samples due to the technical challenge of isolating and analyzing small numbers of cells. The method we describe here has allowed obtaining spatial and temporal tissue-specific transcriptome analysis by coupling LCM with RNA-seq.

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Protocol

As the final product is RNA, take care to avoid contaminating the work with RNases. Wearing gloves is a must. Use diethyl pyrocarbonate (DEPC) -treated water, buffers, etc. Autoclave buffers and bake glassware before use.

1. Tissue fixation

1. Prepare fixative of choice depending on the species and tissue types; for barley seed, use Farmer’s fixative (75% ethanol, 25% glacial acetic acid (v/v)).
2. Chill the fixative on ice prior to harvesting tissues.
3. Collect t.......

Discussion

Many tissue-specific gene expression studies have been limited by hand dissection of samples, which is time-consuming, labor intensive, has a high risk of contamination and can only utilize samples that a human operative is sufficiently dexterous to harvest. LCM is a precise and contact-free technique to collect cells from fixed tissue sections using a mechanically operated laser beam under microscopic visualization.

Good sample preparation is critical for LCM. The process relies upon proper f.......

Materials

Name	Company	Catalog Number	Comments
Acetic acid 100 % ACS/R.	AnalaR NORMAPUR (BioStrategies)	VWRC20104.323
AdhesiveCap 200 opaque	Zeiss	415190-9181-000
Clear base moulds 8 X 10	Leica	3803015
Diethyl pyrocarbonate	Sigma-Aldrich	40718-25ML
High Sensitivity RNA ScreenTape	Agilent	5067-5579
Low­profile disp.blades DB80LS	Leica	14035843489
MembraneSlide 1.0 PEN	Zeiss	415190-9041-000
MessageAmp II aRNA Amplification Kit	Ambion (ThermoFisher)	AMB17515
On-Column DNase I Digestion Set	Sigma-Aldrich	DNASE70
Ovation RNA-Seq System V2	NuGen (Integrated Science)	7102-08
Paraffin (Surgipath Paraplast)	Leica	39601006
PicoPure RNA Isolation Kit	ABI (ThermoFisher)	KIT0214
RNaseZap RNase Decontamination Solution	Ambion (ThermoFisher)	AM9780
Xylene	AnalaR NORMAPUR (BioStrategies)	VWRC28975.360
Leica Benchtop Tissue Processor	Leica Biosystems	TP1020
Leica Heated Paraffin Embedding Module	Leica Biosystems	EG1150H
Leica Cold Plate	Leica Biosystems	EG1150C
Safemate Class 2 Biological Safety Cabinets	LAF Technologies Pty Ltd	Safemate 1.5
Leica Fully Automated Rotary Microtome	Leica Biosystems	RM2265	with PALMRobo v 4.6 software
Zeiss PALM MicroBeam LCM system	Zeiss miscroscopy
TapeStation	Agilent	TapeStation 2200