Name: laser-assisted microdissection (lam) as a tool for transcriptional profiling of individual cell types
Uploaded: 2016-05-10T14:00:00
Description: Watch this Scientific Journal Video about Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types at JoVE.com

We thank Timothy F. Sharbel (IPK Gatersleben) for providing Boechera divaricarpa seeds and Sharon Kessler (University of Oklahoma) for critical reading and proofreading. Work on cell type-specific transcriptome analyses to study gametophyte development and apomixis in UG&#180;s laboratory is supported by the University of Z&#252;rich, by a fellowship of the "Deutsche Forschungsgemeinschaft" and the Marie Curie project IDEAGENA to AS, by grants from the "Staatssekretariat f&#252;r Bildung und Forschung" in the framework of COST action FA0903 (to UG and AS) and the Swiss National Foundation (to UG).

Note: This protocol describes tissue preparation, laser assisted microdissection, and RNA extraction for transcriptional profiling. Always use gloves throughout all steps of the protocol. Study and consider the safety instructions for each chemical used. In particular, keep in mind that Xylol is harmful and can penetrate gloves and that methanol is toxic. For all instruments used, please refer to user manuals accordingly.
1. Removal of RNAse Activity from Glassware and Other Equipment
<ol>
	<li>Prior to use, wra...

<ol>
	<li>Maheshwari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to the Embryology of Angiosperms. , (1950).</li><li>Willemse, M. T. M., Van Went, J. L., Johri, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+female+gametophyte.">The female gametophyte.</a> Embryology of Angiosperms. , 159-196 (1984).</li><li>Koltunow, A. M., Bicknell, R. A., Chaudhury, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apomixis:+Molecular+strategies+for+the+generation+of+genetically+identical+seeds+without+fertilization.">Apomixis: Molecular strategies for the generation of genetically identical seeds without fertilization.</a> Plant Physiol. 108, 1345-1352 (1995).</li><li>Vielle-Calzada, J. P., Crane, C., Stelly, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apomixis+-+the+asexual+revolution.">Apomixis - the asexual revolution.</a> Science. 274, 1322-1323 (1996).</li><li>Grossniklaus, U., Koltunow, A., van Lookeren Campagne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+future+for+apomixis.">A bright future for apomixis.</a> Trends Plant Sci. 3, 415-416 (1998).</li><li>Schmidt, A., Schmid, M. W., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+germline+formation:+molecular+insights+define+common+concepts+and+illustrate+developmental+flexibility+in+apomictic+and+sexual+reproduction.">Plant germline formation: molecular insights define common concepts and illustrate developmental flexibility in apomictic and sexual reproduction.</a> Development. 142, 229-241 (2015).</li><li>Schmidt, 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=Apomictic+and+sexual+germline+development+differ+with+respect+to+cell+cycle,+transcriptional,+hormonal+and+epigenetic+regulation.">Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation.</a> PLOS GENET. 10, e1004476 (2014).</li><li>Okada, T., 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=Enlarging+cells+initiating+apomixis+in+Hieractium+praealtum+transition+to+an+embryo+sac+program+prior+to+entering+mitosis.">Enlarging cells initiating apomixis in Hieractium praealtum transition to an embryo sac program prior to entering mitosis.</a> Plant Physiol. 163, 216-231 (2013).</li><li>Wuest, S. E., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+microdissection+applied+to+floral+tissues.">Laser-assisted microdissection applied to floral tissues.</a> Methods Mol Biol. 1110, 329-344 (2014).</li><li>Wuest, S. E., Schmid, M. W., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+expression+profiling+of+rare+cell+types+as+exemplified+by+its+impact+on+our+understanding+of+female+gametophyte+development.">Cell-specific expression profiling of rare cell types as exemplified by its impact on our understanding of female gametophyte development.</a> Curr Opin Plant Biol. 16 (1), 41-49 (2013).</li><li>Wuest, S. E., 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=Arabidopsis+female+gametophyte+gene+expression+map+reveals+similarities+between+plant+and+animal+gametes.">Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.</a> Curr Biol. 20, 1-7 (2010).</li><li>Cai, S., Lashbrook, C. C. <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+plant+cells+from+tape-transferred+paraffin+sections+promotes+recovery+of+structurally+intact+RNA+for+global+gene+profiling.">Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling.</a> Plant J. 48 (4), 628-637 (2006).</li><li>Schmidt, A., Wuest, S. E., Vijverberg, K., Baroux, C., Kleen, D., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+the+Arabidopsis+megaspore+mother+cell+uncovers+the+importance+of+RNA+helicases+for+plant+germ+line+development.">Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germ line development.</a> PLOS BIOL. 9, e1001155 (2011).</li><li>Schmid, M. W., Schmidt, A., Klostermeier, U. C., Barann, M., Rosenstiel, P., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+powerful+method+for+transcriptional+profiling+of+specific+cell+types+in+eukaryotes:+laser-assisted+microdissection+and+RNA+sequencing.">A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.</a> PLOS ONE. 7, e29685 (2012).</li><li>Mau, M., 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=Hybrid+apomicts+trapped+in+the+ecological+niches+of+their+sexual+ancestors.">Hybrid apomicts trapped in the ecological niches of their sexual ancestors.</a> Proc Natl Acad Sci.U S A. 112 (18), 2357-2365 (2015).</li><li>Aliyu, O. M., Seifert, M., Corral, J. M., Fuchs, J., Sharbel, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copy+number+variation+in+transcriptionally+active+regions+of+sexual+and+apomictic+Boechera.+demonstrates+independently+derived+apomictic+lineages.">Copy number variation in transcriptionally active regions of sexual and apomictic Boechera. demonstrates independently derived apomictic lineages.</a> Plant Cell. 25 (10), 3808-3823 (2013).</li><li>Corral, J. M., 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+conserved+apomixis-specific+polymorphism+is+correlated+with+exclusive+exonuclease+expression+in+premeiotic+ovules+of+apomictic+boechera+species.">A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species.</a> Plant Physiol. 163 (4), 1660-1672 (2013).</li><li>Deeken, R., Ache, P., Kajahn, I., Klinkenberg, J., Bringmann, G., Hedrich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Arabidopsis+thaliana.+phloem+RNAs+provides+a+search+criterion+for+phloem-based+transcripts+hidden+in+complex+datasets+of+microarray+experiments.">Identification of Arabidopsis thaliana. phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments.</a> Plant J. 55, 746-775 (2008).</li><li>Schmid, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genome Scale Quantitative Biology in Arabidopsis thaliana. , 40-104 (2015).</li></ol>

The Protocol is Suitable for Different Cell and Tissue Types
LAM combined with transcriptome analyses by microarrays or RNA-Seq is a valuable tool to gain insights into the specific patterns of gene activity regulating developmental or physiological processes 7-11,13,14. However, the suitability of this method for any given cell type is critically dependent on structural issues. The cell needs to be clearly visible and unambiguously identifiable in the dry sections ...

In transcriptional studies done at the tissue level, the transcriptomes of highly specialized but rare cell types are often masked by the more abundant surrounding cells. An example for such highly specialized cell types are the cells of the female reproductive lineage (germline) in plants. The female germline is specified within developing ovules, the precursors of seeds inside the gynoecium of the flower 1,2. The megaspore mother cell (MMC) is the first cell of the female germline. It undergoes meiosis to form a tetrad of reduced megaspores. Typically, only one of these megaspores survives and divides mitotically without cytokinesis, i.e., in a syncytium. These mitoses are followed by cellularization to form the mature gametophyte, which typically consists of four cell types: three antipodals, two synergid cells, the egg, and the central cell. The egg and central cells are the female gametes that get fertilized by two sperm cells during double fertilization to give rise to the embryo and endosperm of the developing seed 1,2. In the sexual model system Arabidopsis thaliana, only ~ 50 seeds develop per flower while about 50 - 80 seeds develop per flower in the closely related genus Boechera. Thus, the female germline consists of only a few highly specialized cell types, making it an excellent model to study developmental processes, such as cell specification and differentiation.
Moreover, insights into the gene regulatory processes governing plant reproduction can be of applied value. In plants, both sexual and asexual reproduction through seeds (apomixis) can occur. While sexual reproduction generates genetic diversity in a population, apomixis leads to the formation of clonal offspring that is genetically identical to the mother plant. Therefore, apomixis has great potential for applications in agriculture and seed production, as even complex maternal genotypes can be maintained unaltered over several generations 3,4,5. Because apomixis does not naturally occur in any major crop species, the engineering of apomixis in crops is of great interest 3,4,5. However, this long-term goal is difficult to achieve because the underlying genetic and molecular basis of apomixis is not understood in sufficient detail 6.
To gain insights into the transcriptional basis governing apomictic reproduction, cell type-specific transcriptional profiling using laser-assisted microdissection (LAM) and next generation sequencing (NGS) represents a very powerful approach 7,8. LAM has first been established for animal and biomedical research. In the past few years LAM has also been applied to plant biology 6,9,10. In contrast to other methods allowing profiling of individual cell and tissue types, LAM does not require the generation of marker lines 6,9,10. Therefore, it can be applied to any cell or tissue type without prior molecular knowledge. Another advantage of LAM is that it can be applied to any cell type as long as the cell can be recognized in dry sections based on position and/or structural features. LAM has the additional advantage that fixed tissues are used, which prevents changes of the transcriptional profile during processing.
The tissue of interest, e.g., floral tissue, is fixed in a non-crosslinking fixative prior to embedding in paraffin wax. Embedding in paraffin wax can be done manually, following established protocols 9,11. However, the use of an automated tissue processor for dehydration and infiltration with the wax generally results in higher reproducibility in terms of the conservation of RNA quality and tissue morphology. The alternative strategy of embedding tissues in resin has also been successfully used for cell type-specific analyses by LAM 8. However, the use of an automated tissue processor for embedding in wax is very time efficient, as many samples can be processed at once requiring a minimum of hands-on time. While typically no significant loss of RNA quality occurs during fixation and embedding, the preparation of thin sections with the microtome and, in particular, the mounting on the frameslides used for LAM remains a critical step for preservation of RNA quality. This has previously been noted and the use of a tape transfer system has been described to result in better RNA quality at this step 12. However, this adds an additional time-consuming step during preparation of the slides and also requires special equipment. The optimized protocol described below reproducibly produces RNA that is of sufficient quality for transcriptional profiling with GeneCHIPs and Next Generation Sequencing (NGS) approaches 7,11,13,14. In addition, with the laser microdissection microscope used, a high purity of the isolated cell types is routinely produced 7,11,13,14.
The genus Boechera is an excellent model system for studying the key steps of apomictic reproduction. In Boechera, a variety of different sexual and apomictic accessions have been identified and can be used for comparative analyses 15,16,17. In a comparison of cell type-specific transcriptomes of cells from the female germline from sexual Arabidopsis and apomictic Boechera, we identified genes and pathways that are differentially expressed, thereby identifying new aspects of the regulatory processes governing apomixis 7. In addition, this study verified the suitability of LAM for cell type-specific transcriptional analyses of small and rare cell types. We have already used this protocol for the analysis of different cell types in a variety of plant species, but species- and tissue-specific modifications to the protocol may be required in certain cases.

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>1,009,861,000</td>
			<td>absolute EMPROVE Ph Eur,BP,USP</td>
		</tr>
		<tr>
			<td>15 ml falcon centrifuge tubes</td>
			<td>VWR</td>
			<td>62406-200</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Applichem</td>
			<td>A3686,2500</td>
			<td>100% Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ambion Nuclease free water</td>
			<td>life technologies</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>ASP200 S</td>
			<td>Leica</td>
			<td>14048043624</td>
			<td>tissue processor&#160;</td>
		</tr>
		<tr>
			<td>black cardboard</td>
			<td></td>
			<td></td>
			<td>can be purchased in special paper shops</td>
		</tr>
		<tr>
			<td>DNA- and RNAse-free Frame Slides</td>
			<td>Micro Dissect GmbH</td>
			<td></td>
			<td>1,4 &#181;m PET-membrane; can also be purchased from Leica</td>
		</tr>
		<tr>
			<td>Dumond Forceps</td>
			<td>Actimed</td>
			<td>0208-5SPSF-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>exsiccator</td>
			<td>Sigma-Aldrich</td>
			<td>Z354074-1EA</td>
			<td>Nalgene Vaccuum Dessicator or similar equipment</td>
		</tr>
		<tr>
			<td>filter tips 10&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60010</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 1000&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60010</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 20&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60020</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 200&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60200</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>forceps precision</td>
			<td>VWE</td>
			<td>232-1221</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slide holder</td>
			<td>Huber &#38; Co.AG</td>
			<td>10.0540.01</td>
			<td>F&#228;rbek&#228;sten nach Hellendahl</td>
		</tr>
		<tr>
			<td>glass staining trough</td>
			<td>Huber &#38; Co.AG</td>
			<td>10.0570.01</td>
			<td>F&#228;rbekasten</td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Module Leica</td>
			<td>Leica</td>
			<td>Leica EG 1150 H</td>
			<td>blocking station, similar devises are suitable</td>
		</tr>
		<tr>
			<td>Heating and Drying Table</td>
			<td>Medax</td>
			<td>15501</td>
			<td>other models and/or suppliers are suitable</td>
		</tr>
		<tr>
			<td>ice bucket</td>
			<td>VWR ice bucket with lid&#160;</td>
			<td>10146-184</td>
			<td>similar buckets equally suitable</td>
		</tr>
		<tr>
			<td>light table</td>
			<td>UVP An Analytical Jena Company</td>
			<td>TW-26&#160;</td>
			<td>white light transluminator</td>
		</tr>
		<tr>
			<td>microscope slide</td>
			<td>Thermo Scientific</td>
			<td>10143562CE</td>
			<td>cut edges</td>
		</tr>
		<tr>
			<td>microtome blade</td>
			<td>Thermo Scientific</td>
			<td>FEAHS35</td>
			<td>S35 microtome blade disposable</td>
		</tr>
		<tr>
			<td>MMI Cell Cut Plus Instrument</td>
			<td>MMI (Molecular Machines and Industries)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-stick, RNAse free Microfuge tubes, 2ml</td>
			<td>life technologies</td>
			<td>AM12475</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>Roth</td>
			<td>X882.2</td>
			<td>for histology</td>
		</tr>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>life technologies</td>
			<td>KIT0204</td>
			<td>Arcturus PicoPure RNA Isolation Kit</td>
		</tr>
		<tr>
			<td>plastic balancing trays</td>
			<td>Semadeni AG</td>
			<td>2513</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic box</td>
			<td>Semadeni AG</td>
			<td>2971</td>
			<td>Plastikdose PS</td>
		</tr>
		<tr>
			<td>plastic lid for heating plate</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>preparation needle</td>
			<td>VWR</td>
			<td>631-7159</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free microfuge tubes</td>
			<td>life technologies</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse ZAP Decontamination Solution</td>
			<td>life technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-automated Rotary Microtome&#160;</td>
			<td>Leica</td>
			<td>RM2245</td>
			<td>similar devises are equally suitable</td>
		</tr>
		<tr>
			<td>Tissue Loc&#160; Histo Screen Cassettes</td>
			<td>Thermo Scientific</td>
			<td>C-1000_AQ</td>
			<td>similar cassettes of other suppliers are suitable</td>
		</tr>
		<tr>
			<td>Tubes with adhesive lid, without diffusor 500 &#181;l&#160;</td>
			<td>MMI (Molecular Machines and Industries)</td>
			<td>50204</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol (Isomere) ROTIPURAN</td>
			<td>VWR</td>
			<td>4436.2</td>
			<td>min. 99 %, p.a.,ACS, ISO SP</td>
		</tr>
		<tr>
			<td>process embedding cassettes</td>
			<td>Leica</td>
			<td>14039440000</td>
			<td>Leica Jet Cassette I without lid</td>
		</tr>
		<tr>
			<td>Universal Oven</td>
			<td>Memmert</td>
			<td>UF55</td>
			<td>other models and/or suppliers are suitable</td>
		</tr>
	</tbody>
</table>

laser-assisted microdissection (lam) as a tool for transcriptional profiling of individual cell types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Sample Preparation and LAM are done in Consecutive Steps
A number of consecutive steps are required to prepare RNA for transcriptional analysis from selected cell types by LAM (Figure 1). This starts with the harvest of the flowers and immediate fixation to ensure that the RNA population remains unchanged after harvest. The tissue is embedded, sectioned, and mounted on slides. This allows the is...

Watch this Scientific Journal Video about Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types at JoVE.com

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

The overall goal of this protocol for LAM is to isolate specific plant cells for the generation of high-integrity RNA for transcriptional analysis. The focus is on inaccessible cells, like egg cells, in sexual and apomictic boechera species. This method can help answer key questions in the field of plant developmental genetics, such as the transcriptional basis underlying sexual and asexual plant reproduction through seeds.The main advantage of this technique is that certain types specific styles can be perform. Overcoming the effect of masking transcriptions of rare cell types in the analysis of atherogenesis tissues. To begin this procedure, using the blocking station fill a pre-warmed, fresh plastic balancing tray with melted paraffin wax at 56 degrees Celsius.Lift the lid of the paraffin bath and pick up one cassette at a time. Transfer a sample to the 56 degree Celsius liquid paraffin wax in a balancing tray. Proceed quickly to avoid any wax hardening around the sample while handling the cassette.Then, position the sample according to the experimental design using a preparation needle. Repeat the procedure until all the samples are transferred. To prepare thin sections for LAM, on a lighted table, remove the paraffin wax with a sample from the plastic tray.Dissect out a wax block containing the sample, such as a bud or flower, using an RNase-free razor blade. Next, melt a small droplet of parffin wax on the tip of a spatula. And use the hot wax to fix the block with embedded sample on the support.Remove the extra wax surrounding the sample with a razor blade, and make sure the block has a square surface with parallel edges. Then, set the heating plate to 42 degrees Celsius. Safely mount the sample to the microtome.Prepare seven micrometer thick sections for the cells of boechera mature gametophyte. Afterward, transfer the 10 to 15 centimeter long paraffin ribbons to a plastic box with black cardboard at the bottom before positioning them on the LAM slides. To prepare the slides for LAM, place five to ten metal-framed slides on the heating plate with the flat surfaces facing up.Pipette one to two milliliters of RNase-free water on the plastic part of the slides. Using a razor blade, cut the paraffin ribbons in short pieces of about four to five centimeters. Long enough to span the plastic windows of the slides.Then, use a pair of tweezers and a preparation needle to places the paraffin strips parallel to each other on the plastic window of the slides. Carefully lift the slides at one end to remove the water and place them back. Cover the heating plate with a plastic lid without touching the slides, and ensure that the plastic lid allows air exchange.Dry the slides at 42 degrees Celsius overnight. Under a chemical hood, de-wax the slides in xylol two times for 10 minutes each time. Make sure to submerge only the plastic part of the slides and keep the broader end of the metal frame of each slide dry for removal.Allow the slides to dry for 10 minutes in the chemical hood prior to LAM. In this procedure, turn on the computer and the microscope for LAM. Then, start the program steering the microscope and laser.Turn on the laser control box and switch on the laser by pressing the corresponding button on the control box. Next, place a cap into the cap holder. Place the LAM slide on a glass slide from microscopy and ensure that the flat surface of the slide with the tissues attached faces the glass slide.Then, place the slide in the instrument with the glass slide underneath. At last, position the cap holder with the cap in the instrument. Select the 4x objective.In the program move the cap in the up position. Afterward, make a scan of the slide. Search through the slide and identify the structures of interest.Pinpoint and save their positions on the slide to allow returning to the exact position for the cutting step. Then, select an appropriate objective. Verify the laser position by pressing the laser shot button once.Ideally, in a part of the membrane without tissue. With the cap in the down position, use the hand pen tool to mark the borders of the cell type or tissue of interest. For example, the egg cell.Dissect it with the laser by pressing Cut. In order to isolate individual cells, it is critical to clearly identify them in the right sections. Also, the cells should have a diameter of at least eight micrometers to make a cell type specific transcription analysis feasible.Please note that sectioning needs to be repeated if the border around the cell has not been entirely dissected, which can typically be seen on the screen. In this case, it is recommended to set the software to automatically run two repeats of the section as a standard. After that, lift the cap and move to the next save position with a structure of interest and dissect the next cell section until all the cells of interest from one slide are collected.Make sure the cap is positioned in a way that the new section sticks to an empty position on the cap. After the cell types of interest from all the slides have been isolated, remove the slide from the instrument. Place the cap holder in the down position and visually inspect the cap surface.And use the 4x objective to exclude unspecific contaminations of the sample. Then close the cap. And freeze the dry sections at minus 80 degrees Celsius until further use.Here is the schematic drawing of the mature female gametophyte. These are the thin sections through ovules harboring female gametophytes in boechera divaricarpa in which egg cells are clearly identifiable due to the characteristic morphology of the female gametophyte. Similar RNA quality was obtained as analyzed from the egg cell sections compared to the larger tissue areas harvested from the same slides as controls.The RNA quality achieved was suitable for cell type specific RNA sequencing, leading to the identification of 236 genes expressed in the apomictic egg cell but not in the apomictic central cell, synergent cell, or apomictic initial cell. Nor in the cells of the mature sexual gametophyte of arabidopsis thaliana. Other cell and tissue types from different species can be profiled in order to answer additional questions of developmental genetics.Such as the molecular basis of imprinted in the endosperm and embryos of plant seeds. After its development, this technique paves the way for researchers in the field of developmental genetics and plant reproduction to explore the transcriptional basis of apomictic reproduction in the genus Boechera. After watching this video you should have a good understanding of how to obtain good quality RNA for cell type specific transcription analyses using laser-assisted micro-dissection.

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