Name: laser-capture microdissection rna-sequencing for spatial and temporal tissue-specific gene expression analysis in plants
Uploaded: 2020-08-05T14:30:00
Description: Watch this Scientific Journal Video about Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants at JoVE.com

This work was supported by the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008) to JW. M.G.L was supported by a La Trobe University starting grant. We thank the La Trobe Genomics Platform for their support in high-throughput sequencing and data analysis. We thank Associate Professor Matthew Tucker for expert advice on establishing LCM in our lab.

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
<ol>
	<li>Prepare fixative of choice depending on the species and tissue types; for barley seed, use Farmer&#8217;s fixative (75% ethanol, 25% glacial acetic acid (v/v)).</li>
	<li>Chill the fixative on ice prior to harvesting tissues.</li>
	<li>Collect t...

<ol>
	<li>Emmert-Buck, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274 (5289), 998-1001 (1996).</li><li>Alevizos, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+cancer+in+vivo+gene+expression+profiling+assisted+by+laser+capture+microdissection+and+microarray+analysis.">Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis.</a> Oncogene. 20 (43), 6196-6204 (2001).</li><li>Cong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+localization+of+follicular+lymphoma:+description+and+analysis+by+laser+capture+microdissection.">In situ localization of follicular lymphoma: description and analysis by laser capture microdissection.</a> Blood, The Journal of the American Society of Hematology. 99 (9), 3376-3382 (2002).</li><li>Blokhina, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+protocol+for+xylem+tissues+of+woody+plants.">Laser capture microdissection protocol for xylem tissues of woody plants.</a> Frontiers in Plant Science. 7, 1965 (2017).</li><li>Casson, S., Spencer, M., Walker, K., Lindsey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+for+the+analysis+of+gene+expression+during+embryogenesis+of+Arabidopsis.">Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis.</a> The Plant Journal. 42 (1), 111-123 (2005).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LCM-seq+reveals+the+crucial+role+of+LsSOC1+in+heat-promoted+bolting+of+lettuce+(Lactuca+sativa+L.).">LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.).</a> The Plant Journal. 95 (3), 516-528 (2018).</li><li>Jiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+atlas+of+rice+cell+types+uncovers+cellular,+functional+and+developmental+hierarchies.">A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies.</a> Nature Genetics. 41 (2), 258-263 (2009).</li><li>Kivivirta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+laser+microdissection+(LMD)+followed+by+transcriptome+analysis+of+plant+reproductive+tissue+in+phylogenetically+distant+angiosperms.">A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms.</a> Plant Methods. 15 (1), 1-11 (2019).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+dynamics+of+the+maize+leaf+transcriptome.">The developmental dynamics of the maize leaf transcriptome.</a> Nature Genetics. 42 (12), 1060-1067 (2010).</li><li>Liew, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+tissue-specific+regulation+of+transcriptomes+during+barley+(Hordeum+vulgare)+seed+germination.">Temporal tissue-specific regulation of transcriptomes during barley (Hordeum vulgare) seed germination.</a> The Plant Journal. 101 (3), 700-715 (2020).</li><li>Matas, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-and+cell-type+specific+transcriptome+profiling+of+expanding+tomato+fruit+provides+insights+into+metabolic+and+regulatory+specialization+and+cuticle+formation.">Tissue-and cell-type specific transcriptome profiling of expanding tomato fruit provides insights into metabolic and regulatory specialization and cuticle formation.</a> The Plant Cell. 23 (11), 3893-3910 (2011).</li><li>Sakai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+laser-assisted+microdissection+(LAM)+and+RNA-seq+allows+to+perform+a+comprehensive+transcriptomic+analysis+of+epidermal+cells+of+Arabidopsis+embryo.">Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.</a> Plant Methods. 14 (1), 10 (2018).</li><li>Zhan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Sequencing+of+Laser-Capture+Microdissected+compartments+of+the+maize+kernel+identifies+regulatory+modules+associated+with+endosperm+cell+differentiation.">RNA Sequencing of Laser-Capture Microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation.</a> The Plant Cell. 27 (3), 513-531 (2015).</li><li>Hwang, B., Lee, J. H., Bang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-14 (2018).</li><li>Zeb, Q., Wang, C., Shafiq, S., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Cell Omics. , 101-135 (2019).</li><li>Deal, R. B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nature Protocols. 6 (1), 56 (2011).</li><li>Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+mRNA+purification+by+translating+ribosome+affinity+purification+(TRAP).">Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).</a> Nature Protocols. 9 (6), 1282 (2014).</li><li>Bevilacqua, C., Ducos, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection:+A+powerful+tool+for+genomics+at+cell+level.">Laser microdissection: A powerful tool for genomics at cell level.</a> Molecular Aspects of Medicine. 59, 5-27 (2018).</li><li>Nelson, T., Tausta, S. L., Gandotra, N., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+plant+tissue:+what+you+see+is+what+you+get.">Laser microdissection of plant tissue: what you see is what you get.</a> Annual Reviews in Plant Biology. 57, 181-201 (2006).</li><li>Day, R. C., Grossniklaus, U., Macknight, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Be+more+specific!+Laser-assisted+microdissection+of+plant+cells.">Be more specific! Laser-assisted microdissection of plant cells.</a> Trends in Plant Science. 10 (8), 397-406 (2005).</li><li>Takahashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+obtaining+high+quality+RNA+from+paraffin+sections+of+plant+tissues+by+laser+microdissection.">A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection.</a> Journal of Plant Research. 123 (6), 807-813 (2010).</li><li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Molecular Biology. 7 (1), 3 (2006).</li><li>Ferreira, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+mRNA+amplification+approach+for+RNAseq+from+limited+amount+of+RNA.">Linear mRNA amplification approach for RNAseq from limited amount of RNA.</a> Gene. 564 (2), 220-227 (2015).</li><li>Schneider, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+T7+RNA+polymerase+based+in+vitro+linear+RNA+amplification+for+use+in+microarray+experiments.">Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments.</a> BMC Genomics. 5 (1), 29 (2004).</li><li>Shanker, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+commercially+available+RNA+amplification+kits+for+RNA+sequencing+using+very+low+input+amounts+of+total+RNA.">Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.</a> Journal of Biomolecular Techniques. 26 (1), 4 (2015).</li><li>Bhattacherjee, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+fluorescently+labeled+embryonic+cranial+neural+crest+cells.">Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells.</a> Genesis. 39 (1), 58-64 (2004).</li><li>Cl&#233;ment-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+RNA+during+laser+capture+microdissection+by+performing+experiments+under+argon+atmosphere+or+using+ethanol+as+a+solvent+in+staining+solutions.">Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.</a> RNA. 14 (12), 2698-2704 (2008).</li><li>Blokhina, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenchymal+Cells+Contribute+to+Lignification+of+Tracheids+in+Developing+Xylem+of+Norway+Spruce.">Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce.</a> Plant Physiology. 181 (4), 1552-1572 (2019).</li><li>Schad, M., Lipton, M. S., Giavalisco, P., Smith, R. D., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two-dimensional+electrophoresis+and+liquid+chromatography-tandem+mass+spectrometry+for+tissue-specific+protein+profiling+of+laser-microdissected+plant+samples.">Evaluation of two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples.</a> Electrophoresis. 26 (14), 2729-2738 (2005).</li><li>Schad, M., Mungur, R., Fiehn, O., Kehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling+of+laser+microdissected+vascular+bundles+of+Arabidopsis+thaliana.">Metabolic profiling of laser microdissected vascular bundles of Arabidopsis thaliana.</a> Plant Methods. 1 (1), 2 (2005).</li><li>Latrasse, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quest+for+epigenetic+regulation+underlying+unisexual+flower+development+in+Cucumis+melo.">The quest for epigenetic regulation underlying unisexual flower development in Cucumis melo.</a> Epigenetics &amp; Chromatin. 10 (1), 22 (2017).</li><li>Turco, G. 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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...

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.
LCM was developed two decades ago by Emmert-Buck and colleagues1. The technique enabled researchers to precisely isolate single-cells or clusters of cells from their environment using direct microscopic visualization and manipulation with a narrow beam laser1. Since then the method has been widely used in cancer biology and pathology2,3. Many plant research groups have also adapted LCM for the use with different plant species and different tissue types4,5,6,7,8,9,10,11. Recently, several papers have also used LCM on eudicot and monocot seeds to study embryo, endosperms and other seed structures during seed development and germination10,12,13. Most of the other commonly used single-cell isolation methods such as micro-pipetting, cell sorting, magnetic separation and microfluidic platforms depend on the enzymatic digestion or mechanical homogenization to dissociate cells. This may perturb gene expression, introducing technical artefacts that confound data interpretation14,15. These methods also require previous knowledge of marker genes for each cell type to relate the dissociated cells to their spatial location and true cell-type. A further group of techniques depends on affinity-based isolation of subcellular structures instead of whole cells, for example INTACT (Isolation of Nuclei Tagged in Cell Types) and TRAP (Translating Ribosome Affinity Purification)16,17. However, affinity labeling and purification of nuclei or ribosomes are technically challenging in plant species that do not have well-established transformation protocols. LCM takes advantage of quick tissue fixation to preserve transcript levels and conventional histological identification by direct visualization of cells within their normal tissue/organ context, which allows discrete cells to be isolated in a short period of time18,19.
The protocol presented here is an optimized method for the isolation of specific cells or cell types from the tissue sections of cereal seeds, which can be applied to most of the cells that can be histologically identified. LCM provides a contact-free method of cell isolation, greatly reducing contamination and increasing integrity of recovered RNA. Furthermore, the method illustrates the power of LCM on large-scale genome wide studies starting with small quantities of biological materials. We also describe linear amplification of RNA for generating sufficient input material for downstream transcript/transcriptome analyses.
There are ten main steps in this LCM RNA-seq protocol for spatial and temporal tissue-specific transcriptomes, including fixation of tissue samples, dehydration, paraffin infiltration, embedding, sectioning, LCM, RNA extraction, RNA amplification, RNA quantification and qRT-PCR and/or RNA-seq (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61517/61517fig01.jpg" /> 
Figure 1: Flowchart of LCM followed by RNA-seq or qRT-PCR.&#160;LCM is a spatially precise and contact-free technique to collect cells from fixed tissue sections using a laser beam under microscopic visualization. The process starts with fixation of tissue samples, followed by dehydration using a gradient series of ethanol and xylene, and finished with paraffin infiltration. The process can be fully automated by using a tissue processor. Once the tissue is infiltrated with paraffin, it is embedded in a mold with molten paraffin using an embedding station. Sectioning is carried out using microtome set to the desired thickness. Slides are prepared and LCM conducted immediately before RNA is to be extracted from captured cells. RNA extraction is followed directly by two rounds of RNA amplification prior to qRT-PCR and/or RNA-seq. <a href="https://www.jove.com/files/ftp_upload/61517/61517fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Acetic acid 100 % ACS/R.</td>
			<td>AnalaR NORMAPUR (BioStrategies)</td>
			<td>VWRC20104.323</td>
			<td></td>
		</tr>
		<tr>
			<td>AdhesiveCap 200 opaque</td>
			<td>Zeiss</td>
			<td>415190-9181-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear base moulds 8 X 10</td>
			<td>Leica</td>
			<td>3803015</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>40718-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity RNA ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5579</td>
			<td></td>
		</tr>
		<tr>
			<td>Low&#173;profile disp.blades DB80LS</td>
			<td>Leica</td>
			<td>14035843489</td>
			<td></td>
		</tr>
		<tr>
			<td>MembraneSlide 1.0 PEN</td>
			<td>Zeiss</td>
			<td>415190-9041-000</td>
			<td></td>
		</tr>
		<tr>
			<td>MessageAmp II aRNA Amplification Kit</td>
			<td>Ambion (ThermoFisher)</td>
			<td>AMB17515</td>
			<td></td>
		</tr>
		<tr>
			<td>On-Column DNase I Digestion Set</td>
			<td>Sigma-Aldrich</td>
			<td>DNASE70</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation RNA-Seq System V2</td>
			<td>NuGen (Integrated Science)</td>
			<td>7102-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin (Surgipath Paraplast)</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>ABI (ThermoFisher)</td>
			<td>KIT0214</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Ambion (ThermoFisher)</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>AnalaR NORMAPUR (BioStrategies)</td>
			<td>VWRC28975.360</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Benchtop Tissue Processor</td>
			<td>Leica Biosystems</td>
			<td>TP1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Heated Paraffin Embedding Module</td>
			<td>Leica Biosystems</td>
			<td>EG1150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Cold Plate</td>
			<td>Leica Biosystems</td>
			<td>EG1150C</td>
			<td></td>
		</tr>
		<tr>
			<td>Safemate Class 2 Biological Safety Cabinets</td>
			<td>LAF Technologies Pty Ltd</td>
			<td>Safemate 1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Fully Automated Rotary Microtome</td>
			<td>Leica Biosystems</td>
			<td>RM2265</td>
			<td>with PALMRobo v 4.6 software</td>
		</tr>
		<tr>
			<td>Zeiss PALM MicroBeam LCM system</td>
			<td>Zeiss miscroscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation</td>
			<td>Agilent</td>
			<td>TapeStation 2200</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-capture microdissection rna-sequencing for spatial and temporal tissue-specific gene expression analysis in plants

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.

We generated spatial and temporal tissue-specific transcriptomes from barley seeds during germination using our LCM RNA-seq protocol10. The study was carried out by applying LCM RNA-seq to small number of cells from three embryo organs (plumule, radicle tip, scutellum) every 8 h over a 48 h time course during germination (0-48 h, 7 time points) (Figure 2A,B).
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Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

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.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

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.For barley seed, cut the seed samples in half before submerging the samples into at least a 10 X volume of ice cold fixative. Use vacuum infiltration to accelerate the penetration of the fixative. The tissue should sink by the end of the infiltration, then refresh the fixative for an overnight incubation and transfer the samples into cassettes for tissue processing.Place the tissue loaded cassettes into the metal basket of an automatic processor and attach the metal basket to its holder above chamber one. Set the program on the control panels of the tissue processor and press start to begin the automatic processing program. The system will dehydrate the samples by dipping the cassettes into a gradient series of ethanol for 90 minutes per concentration as indicated.After the last ethanol immersion, the system will submerge the samples in xylene gradients for 90 minutes per solution as indicated. Followed by 290 minute immersions in molten paraffin at 55 to 60 degrees Celsius. The next morning, remove the paraffin infiltrated cassettes from the tissue processor and proceed to the paraffin embedding.The next morning, use fine forceps to transfer the process samples into suitably sized molds and add molten paraffin over each sample. For barley seeds, orient the samples in the paraffin longitudinally to the cutting direction to facilitate the acquisition of longitudinal sections. Place a clean cassette onto the mold and ensure that the entire cassette is fully covered with a sufficient volume of paraffin to secure the sample to the cassette.Then place the mold onto a cold plate for 10 to 20 minutes until the paraffin is set before releasing the block from the mold. To prepare a PEN Membrane Slides, submerge the slides in RNS deactivating solution for three seconds followed by two brief washes and DEPC treated water. Then drive the slides in a 370 degrees Celsius incubator to remove any leftover solution and UV treat the slides in a laminar flow cabinet for 30 minutes to enhance their paraffin not hazelnuts.For a sample tissue sectioning place the paraffin blocks on the cold plate and fill the water bath with the DEPC treated water, then warm to 42 degrees Celsius. Trim blocks to the depth of the region of interest and section the paraffin blocks to a six to 10 micron thickness. A well sectioned block will form a ribbon at the edge of the blade.Use a fine tweezer to gently transfer ribbons from the microtome to the water bath, taking care that the ribbon lays flat on the surface of the water. To collect the sections, holding a slide at a 45 degree angle, use an upward motion to lift a ribbon out of the water onto the slide and use a lint free tissue to carefully remove the excess water. When all of the sections have been collected, wash the slides with 320 second washes in xylene followed by 230 seconds washes in 100%ethanol and 230 second washes in 70%ethanol.To microdissect cells of interest from the deparraffinized and dry tissue sections, load slides on the three LCM microscope slots and load collection tube into the available slots. Move the stage to locate the region of the sample that needs to be cut and cut a blank segment free of tissue on the membrane slide. To optimize the cutting speed and the cutting in laser pressure catapulting energy and focus.Use the drawing tools to outline the area of interest in the tissue and use the optimized parameters and the Robo LPC function to catapult cells into the adhesive caps. Select flag from the elements list to use the flag tool to mark the regions of interest. Check the cap check button to inspect the adhesive cap, to confirm that the samples have been captured.Typically 10 to 15 sections per cap are required for RNA extraction. Immediately after all of the samples have been acquired, proceed immediately to RNA extraction to avoid RNA degradation. After RNA extraction and RNA amplification use an automated electrophoresis system, according to the manufacturer's instructions to quantify and qualify the antisense RNA.In this study, LCM, RNA-sequencing was applied to a small number of cells from three embryo organs, every eight hours over a 48 hour time course during germination. It's important to adjust the cutting parameters correctly to allow the tissue of interest to be precisely cut and dislodged from the surrounding tissue without burning the edge of the selected area. Ramification of the total RNA before RNA amplification allows the detection of distinct electrophoretic bands and fluorescent peaks of 18 and 28S ribosomal subunits in good quality RNA samples.Successfully synthesized antisense RNA will exhibit a unimodal symmetrical size distribution from 100 to 1000 nucleotides with a peak around 300 nucleotides after two rounds of amplification. Multi-dimensional scale plotting of genes expressed in the different tissues over 48 hours of germination, illustrates a greater similarity between samples of a single tissue than between samples from the same time point, but different tissues. The number of differentially expressed genes increases progressively over the course of germination in each tissue relative to the zero hour time point of the tissue.In this representative analysis, 25%of plumule, 34%of radicle and 41%of scutellum differentially expressed genes were found to be exclusively expressed within each tissue. It is important to correctly adjust the cutting parameters for a precise excision and dislodging of the selector area from the surrounding tissue without burning the edge of the selected region. With improved chromatin and protein purification methods, and enhance mass spectrometry instrument, LCM can be used for cell type specific epigenomic and proteomics studies implants.

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