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).

<ol><li>Maheshwari, P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMaheshwari%2C+P+%22An+Introduction+to+the+Embryology+of+Angiosperms%22">An Introduction to the Embryology of Angiosperms</a>. , McGraw-Hill. New York. (1950).</li>
<li>Willemse, M. T. M., Van Went, J. L. The female gametophyte. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWillemse%2C+MTM+%22Embryology+of+Angiosperms%22">Embryology of Angiosperms</a>. Johri, B. M. , Springer-Verlag. Berlin. 159-196 (1984).</li>
<li>Koltunow, A. M., Bicknell, R. A., Chaudhury, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apomixis%3A+Molecular+strategies+for+the+generation+of+genetically+identical+seeds+without+fertilization%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">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/pubmed?term=((Apomixis+-+the+asexual+revolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((A+bright+future+for+apomixis%5BTitle%5D)+AND+%22Trends+Plant+Sci%22%5BJournal%5D)">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/pubmed?term=((Plant+germline+formation%3A+molecular+insights+define+common+concepts+and+illustrate+developmental+flexibility+in+apomictic+and+sexual+reproduction%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Apomictic+and+sexual+germline+development+differ+with+respect+to+cell+cycle%2C+transcriptional%2C+hormonal+and+epigenetic+regulation%5BTitle%5D)+AND+%22PLOS+GENET%22%5BJournal%5D)">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/pubmed?term=((Enlarging+cells+initiating+apomixis+in+Hieractium+praealtum+transition+to+an+embryo+sac+program+prior+to+entering+mitosis%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">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/pubmed?term=((Laser-assisted+microdissection+applied+to+floral+tissues%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Cell-specific+expression+profiling+of+rare+cell+types+as+exemplified+by+its+impact+on+our+understanding+of+female+gametophyte+development%5BTitle%5D)+AND+%22Curr+Opin+Plant+Biol%22%5BJournal%5D)">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/pubmed?term=((Arabidopsis+female+gametophyte+gene+expression+map+reveals+similarities+between+plant+and+animal+gametes%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">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/pubmed?term=((Laser+capture+microdissection+of+plant+cells+from+tape-transferred+paraffin+sections+promotes+recovery+of+structurally+intact+RNA+for+global+gene+profiling%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">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/pubmed?term=((Transcriptome+analysis+of+the+Arabidopsis+megaspore+mother+cell+uncovers+the+importance+of+RNA+helicases+for+plant+germ+line+development%5BTitle%5D)+AND+%22PLOS+BIOL%22%5BJournal%5D)">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/pubmed?term=((A+powerful+method+for+transcriptional+profiling+of+specific+cell+types+in+eukaryotes%3A+laser-assisted+microdissection+and+RNA+sequencing%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">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/pubmed?term=((Hybrid+apomicts+trapped+in+the+ecological+niches+of+their+sexual+ancestors%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci.U+S+A%22%5BJournal%5D)">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/pubmed?term=((Copy+number+variation+in+transcriptionally+active+regions+of+sexual+and+apomictic+Boechera.+demonstrates+independently+derived+apomictic+lineages%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((A+conserved+apomixis-specific+polymorphism+is+correlated+with+exclusive+exonuclease+expression+in+premeiotic+ovules+of+apomictic+boechera+species%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">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/pubmed?term=((Identification+of+Arabidopsis+thaliana.+phloem+RNAs+provides+a+search+criterion+for+phloem-based+transcripts+hidden+in+complex+datasets+of+microarray+experiments%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22Genome+Scale+Quantitative+Biology+in+Arabidopsis+thaliana%22">Genome Scale Quantitative Biology in Arabidopsis thaliana</a>. , University of Zürich. PhD Thesis 40-104 (2015).</li>
</ol>

The authors have nothing to disclose.

Das Protokoll eignet sich für verschiedene Zell- und Gewebetypen LAM mit Transkriptom kombiniert Analysen von Microarrays oder RNA-Seq ist ein wertvolles Werkzeug Einblicke in die spezifischen Muster der Gen - Aktivität zu gewinnen , Entwicklungs- oder physiologische Prozesse regulieren 7-11,13,14. Jedoch ist die Eignung dieses Verfahren für jeden gegebenen Zelltyp kritisch abhängig von strukturellen Problemen. Die Zelle muss gut sichtbar und ...

In Transkriptions Studien an der Gewebeebene getan werden die Transkriptom von hoch spezialisierten, aber seltene Zelltypen durch die reichlicher umgebenden Zellen oft maskiert. Ein Beispiel für eine solche hochspezialisierte Zelltypen sind die Zellen der weiblichen Fortpflanzungs lineage (Keimbahn) in Pflanzen. Die weibliche Keimbahn ist in der Entwicklung Ovula angegeben, die Vorläufer von Samen im Inneren der gynoecium der Blume 1,2. Der Megasporenmutterzelle (MMC) ist die erste Zelle des weiblichen Keimbahn. Es erfährt Meiose ein tetrad reduzierter Megasporen zu bilden. Typischerweise überlebt nur eine dieser Megasporen und teilt sich mitotisch ohne Zytokinese, dh in einem Syncytium. Diese Mitosen werden von Zellularisierung folgte der reifen gametophyte zu bilden, die in der Regel besteht aus vier Zelltypen: drei antipodals, zwei synergid Zellen, das Ei und die zentrale Zelle. Das Ei und Zentralzellen sind die weiblichen Gameten, die von zwei Samenzellen während dou befruchtetble Fertilisation die zu den Embryo und Endosperm des entwickelnden Samen 1,2 zu ergeben. Im sexuellen Modellsystem Arabidopsis thaliana, nur ~ 50 Samen pro Blüte entwickeln , während etwa 50 bis 80 Samen pro Blume in der nahe verwandten Gattung Boechera entwickeln. Somit besteht die weiblichen Keimbahn von nur wenigen hochspezialisierte Zelltypen, ist es ein ausgezeichnetes Modell macht Entwicklungsprozesse, wie Zell Beschreibung und Differenzierung zu studieren. Darüber hinaus Einblicke in die Gen-regulatorischen Prozesse Pflanzenreproduktion regeln kann der angewandten Wert sein. In Pflanzen können sowohl sexuelle und asexuelle Vermehrung durch Samen (Apomixis) auftreten. Während der sexuellen Reproduktion genetische Vielfalt in einer Population erzeugt, Apomixis führt zur Bildung von klonale Nachkommenschaft, die genetisch identisch mit der Mutterpflanze ist. Daher hat apomixis ein großes Potenzial für Anwendungen in der Landwirtschaft und der Saatgutproduktion, als auch komplexe mütterliche Genotypen3,4,5 beibehalten unverändert über mehrere Generationen werden. Da apomixis natürlich nicht in allen wichtigen Kulturarten auftreten, ist das Engineering von Apomixis in Kulturen von großem Interesse 3,4,5. Doch dieses langfristige Ziel ist schwer zu erreichen , da die zugrunde liegenden genetischen und molekularen Grundlagen von apomixis nicht ausreichend detailliert 6 verstanden wird. Um einen Einblick in die Transkriptions Basis gewinnen regeln apomictic Reproduktion, zelltypspezifischen Transkriptionsprofilierung mit lasergestützten Mikrodissektion (LAM) und Next Generation Sequencing (NGS) stellt einen sehr leistungsfähigen Ansatz 7,8. LAM hat zunächst für Tier und der biomedizinischen Forschung etabliert. In den letzten Jahren hat LAM auch auf Pflanzenbiologie 6,9,10 angewendet. Im Gegensatz zu anderen Verfahren ermöglicht Profilierung einzelner Zell- und Gewebetypen, LAM erfordert nicht die Erzeugung von Markierungslinien 6,9,10. Daher kann es sein, applog auf eine beliebige Zelle oder Gewebetyp ohne vorherige molekulare Wissen. Ein weiterer Vorteil der LAM ist, dass es kann so lange in jedem Zelltyp angewendet werden, wie die Zelle kann in Trockenabschnitten basierend auf Position und / oder Strukturmerkmale erkannt werden. LAM hat den zusätzlichen Vorteil, dass fixierten Geweben verwendet werden, die bei der Verarbeitung Veränderungen des Transkriptionsprofils verhindert. Das Gewebe von Interesse, beispielsweise Blütengewebe wird in einem nicht vernetzenden Fixiermittel fixiert , bevor sie in Paraffin eingebettet wird . Einbettung in Paraffinwachs kann manuell durchgeführt werden, nach etablierten Protokollen 9,11. Jedoch führt die Verwendung eines automatischen Gewebeprozessor zur Dehydratisierung und Infiltration mit dem Wachs in der Regel höhere Reproduzierbarkeit in Bezug auf die Erhaltung der RNA-Qualität und Gewebemorphologie. Die alternative Strategie von Geweben in Harz einzubetten wurde auch für Zelltyp-spezifischen Analysen von LAM 8 erfolgreich verwendet. Jedoch ist die Verwendung eines automated Gewebeprozessor in Wachs zur Einbettung ist sehr zeitsparend, da viele Proben einmal verarbeitet werden können, auf ein Minimum von hands-on Zeit erfordern. Während typischerweise während der Fixierung und Einbettung keinen signifikanten Verlust an RNA-Qualität erfolgt, bleibt die Herstellung von Dünnschnitten mit dem Mikrotom und insbesondere auf den Frameslides die Halterung für LAM verwendet ein kritischer Schritt für die Konservierung von RNA-Qualität. Dies wurde zuvor festgestellt , und die Verwendung eines Bandübertragungssystem hat bei diesem Schritt in einer besseren Qualität RNA resultieren 12 beschrieben. Dies jedoch fügt einen zusätzlichen zeitaufwendigen Schritt bei der Herstellung der Folien und erfordert auch spezielle Ausrüstung. Das optimierte Protokoll unter reproduzierbar beschrieben produziert RNA , die für die Transkriptionsprofilierung mit Genchips und Next Generation Sequencing (NGS) nähert sich 7,11,13,14 von ausreichender Qualität ist. Darüber hinaus verwendet, um mit dem Laser Mikrodissektion Mikroskop, eine hohe Reinheit der isolierten Zelltypen ist routinely produziert 7,11,13,14. Die Gattung Boechera ist ein ausgezeichnetes Modellsystem für die wichtigsten Schritte von apomictic Wiedergabe zu studieren. In Boechera, eine Vielzahl unterschiedlicher sexueller und apomictic Beitritte wurden identifiziert und können für Vergleichsanalysen verwendet werden 15,16,17. In einem Vergleich von zelltypspezifischen Transkriptomen von Zellen aus der weiblichen Keimbahn von sexuellen Arabidopsis und apomictic Boechera identifizierten wir Gene und Wege , die differentiell exprimiert werden, um dadurch neue Aspekte der regulatorischen Prozesse identifizieren Apomixis 7 regelt. Darüber hinaus überprüft diese Studie die Eignung von LAM für zelltypspezifischen Transkriptionsanalysen von kleinen und seltenen Zelltypen. Wir haben bereits dieses Protokoll für die Analyse verschiedener Zelltypen in einer Vielzahl von Pflanzenarten verwendet, aber arten und gewebespezifische Modifikationen am Protokoll kann in bestimmten Fällen erforderlich sein.

<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&amp;nbsp;</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 &amp;micro;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&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60010</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 1,000&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60010</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 20&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60020</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 200&amp;nbsp;&amp;micro;l</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 &amp;amp; Co.AG</td><td>10.0540.01</td><td>F&amp;auml;rbek&amp;auml;sten nach Hellendahl</td></tr><tr><td>glass staining trough</td><td>Huber &amp;amp; Co.AG</td><td>10.0570.01</td><td>F&amp;auml;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&amp;nbsp;</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&amp;nbsp;</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, 2 ml</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&amp;nbsp;</td><td>Leica</td><td>RM2245</td><td>similar devices are equally suitable</td></tr><tr><td>Tissue Loc 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 &amp;micro;l&amp;nbsp;</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.

Probenvorbereitung und LAM sind in aufeinanderfolgenden Schritten durchgeführt Eine Anzahl von aufeinanderfolgenden Schritte sind erforderlich RNA für die Transkriptionsanalyse von ausgewählten Zelltypen von LAM (Abbildung 1) herzustellen. Dies beginnt mit der Ernte der Blüten und sofortige Fixierung, um sicherzustellen, dass die RNA-Population nach der Ernte unverändert bleibt. Das Geweb...

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

Lasergestützte Mikrodissektion (LAM) als Werkzeug für die Transkriptionsprofilierung einzelner Zelltypen

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.

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

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Biology

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

9.3K Aufrufe.  University of Zürich & Zürich-Basel Plant Science Center. 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. 

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

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Transkriptom-Analyse von Einzelzellen

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Forschung

Neurowissenschaften

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non-Laser Capture Microscopy Ansatz für die Mikrodissektion von Discrete-Maus Hirnregionen für Gesamt-RNA Isolierung und Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Kombination von Laser Capture Microdissection und Microfluidic qPCR zur Analyse von Transkriptionsprofilen einzelner Zellen: Ein systembiologischer Ansatz zur Opioidabhängigkeit

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular ...

Genetik

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Capture Mikrodissektion von Angereichert Populationen von Neuronen oder Single Neurons zur Genexpressionsanalyse Nach Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser Capture Mikrodissektion von Maus embryonalen Knorpel und Knochen für Die Genexpressionsanalyse

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Entwicklungsbiologie

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Laser Mikrodissektion Angewandt auf Gene Expression Profiling von Untergruppe von Zellen aus dem Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Biologie

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Mikrodissektion - Eine Demonstration der Isolation der einzelnen Dopamin-Neuronen und die gesamte ventrale Tegmentum

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-Mikrodissektion für Menschen Prostataepithel für die RNA-Analyse

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Medizin

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

RNA-Isolierung aus zellspezifische Subpopulationen Mit Laser-Mikrodissektion In Verbindung mit Rapid-Immunmarkierung

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Laser-Mikrodissektion für speziesunabhängige Einzelgewebeanwendungen

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...