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 ...

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Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...