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://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 authors have nothing to disclose.

의정서는 다른 세포와 조직에 적합하다 사체와 함께 LAM은 마이크로 어레이 또는으로 분석 RNA-서열은 개발 또는 생리 학적 과정 7-11,13,14을 조절하는 유전자 활동의 특정 패턴에 대한 통찰력을 얻을 수있는 유용한 도구입니다. 그러나, 특정 세포 타입에 대해,이 방법의 적합성 구조적 문제에 비판적으로 의존한다. 셀은 명확하게 볼 수 있고 LAM에 사용되는 ...

조직 수준에서 수행 전사 연구에서 매우 전문화하지만 희귀 세포 유형의 전 사체는 종종 더 풍부 주변 세포에 의해 마스크된다. 이러한 고도로 전문화 된 세포 유형에 대한 예는 식물에서 여성의 생식 계통 (생식 세포)의 세포이다. 여성 생식 계열은 꽃 1, 2의 gynoecium 내부 개발 난자 내 씨의 전구체를 지정합니다. megaspore 모 세포 (MMC)는 여성 생식계의 첫번째 셀이다. 그것은 감​​소 megaspores의 테트라을 형성하는 감수 분열을 거친다. syncytium에, 즉, 일반적으로 이러한 megaspores 중 하나만이 살아과 세포질 분열없이 mitotically 나눕니다. 세 antipodals 두 synergid 세포, 난자, 중앙 셀 : 유사 분열이 전형적으로는 네 종류의 세포로 구성된 성숙 배우체를 형성 cellularization 뒤 따른다. 계란과 중앙 세포는 DOU 동안 두 개의 정자 세포에 의해 수정 된 얻을 여성의 생식있다BLE 시비 현상 시드 1,2 배아 및 배유을 야기한다. 80 씨앗 밀접한 관련 속 Boechera에 꽃 당 개발 - 약 50 동안 성적 모델 시스템 애기 장대에서 만 ~ 50 씨앗은 꽃마다 개발한다. 따라서 암컷 생식선은 휴대 사양과 분화 발달 과정 등을 연구하는 우수한 모델을 만드는 몇 매우 특별한 종류의 세포로 구성된다. 또한, 식물의 재생을 지배하는 유전자 조절 과정에 대한 통찰력은 응용 가치가있을 수 있습니다. 식물, 씨앗 (무수정 생식)을 통해 모두 성적과 무성 생식가 발생할 수 있습니다. 유성 생식은 인구의 유전 적 다양성을 생성하는 동안, 어머니 공장에 유 전적으로 동일 클론 자손의 형성에 리드를 무수정 생식. 따라서, 무수정 생식은 복잡한 산모의 유전자형이 할 수있는, 농업과 종자 생산에 응용 프로그램에 대한 큰 잠재력을 가지고여러 세대 3,4,5를 통해 변경되지 않은 유지 될 수있다. 무수정 생식 자연스럽게 주요 작물에 발생하지 않기 때문에, 작물의 무수정 생식의 공학은 큰 관심의 3,4,5이다. 그러나, 이러한 장기 목표 무수정 생식의 근본적인 유전 및 분자 기초는 충분히 상세히 6에서 이해되지 않기 때문에 달성하기 어렵다. 레이저를 이용한 미세 절제 (LAM) 및 차세대 시퀀싱 (NGS)를 사용하여 apomictic 재생, 세포 유형 특정 전사 프로파일 링을 지배하는 전사 기준에 대한 통찰력을 확보하는 것은 매우 강력한 방법 7,8를 나타냅니다. LAM 먼저 동물과 생물 의학 연구를 위해 설립되었습니다. 지난 몇 년 동안 LAM은 식물학 6,9,10 적용되었다. 개별적인 세포 및 조직 유형의 프로파일을 허용하는 다른 방법들과 대조적으로, LAM 마커 라인 6,9,10의 생성을 필요로하지 않는다. 따라서, 애플리케이션이 될 수있다이전 분자 지식없이 세포 또는 조직 유형에 거짓말. LAM의 또 다른 장점은 셀이 위치 및 / 또는 구조적 특성에 기초하여 건조 부에서 인식 될 수있는 것이 긴 어떠한 세포 유형에 적용 할 수 있다는 것이다. LAM은 처리 중에 전사 프로파일의 변화를 방지 고정 조직을 사용하는 추가적인 장점을 갖는다. 관심, 예를 들어, 꽃 조직의 조직은, 이전에 파라핀 왁스에 매립 비 가교 고정액에 고정되어있다. 파라핀 왁스에 포함 설립 프로토콜 9,11 다음, 수동으로 수행 할 수 있습니다. 그러나, 왁스의 침투와 탈수를위한 자동 조직 프로세서의 사용은 일반적으로 RNA 품질 및 조직 형태의 보전의 관점에서 높은 재현성을 초래한다. 수지 조직을 포함하는 대안의 전략은 성공적 LAM 8 세포 유형 특정한 분석을 위해 사용되어왔다. 그러나, 자동 게이트의 사용많은 샘플 일단 시간의 실질적인 최소 요구 처리 될 수있는 왁스 매립 ated 조직 프로세서는 시간이 매우 효과적이다. RNA의 질 전형적으로 상당한 손실이 고정 및 매립시 발생하는 동안, 마이크로톰 얇은 부분의 제조 및 특히는 LAM에 사용 frameslides에 장착하면 RNA 품질의 보존을위한 중요한 단계 남아있다. 이는 앞서 언급 한 내용 및 테이프 이송 시스템의 이용이 단계 (12)에서 더 RNA 품질 결과를 설명 하였다. 그러나,이 슬라이드의 제조시 추가의 시간 소모적 인 단계를 추가하고 또한 특별한 장치를 필요로한다. 설명 최적화 된 프로토콜은 재현성 GeneCHIPs 및 차세대 시퀀싱 (NGS)이 7,11,13,14 접근과 전사 프로파일에 충분한 품질의 RNA를 생성합니다. 또한, 사용되는 레이저 미세 절제 현미경 단리 세포 유형의 고순도 ROUT은inely은 7,11,13,14을 생산했다. 속 Boechera는 apomictic 재생의 주요 단계를 공부를위한 훌륭한 모델 시스템입니다. Boechera에서, 상이한 성적 apomictic 가입 나 다양한 식별되고, 비교에 사용될 수 15, 16, 17은 해석한다. 성적 애기 장대와 apomictic Boechera에서 여성의 배아에서 세포의 세포 유형 별 전 사체의 비교에서, 우리는 이에 7 무수정 생식에 적용되는 규제 프로세스의 새로운 측면을 식별 차별적으로 발현되는 유전자와 경로를 확인했다. 또한,이 연구는 세포 유형 특이 적 전사 대 LAM의 적합성 작은 ​​희귀 세포 유형의 분석을 확인 하였다. 이미 식물 종의 다양한 상이한 세포 유형의 분석이 프로토콜을 사용하지만, 종 - 상기 프로토콜 조직 - 특이 적 변형은 특정한 경우에 요구 될 수있다.

<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.

샘플 준비 및 LAM은 연속 단계에서 완료 연속적인 단계들의 수는 LAM (도 1)에 의해 선택된 세포 유형에서 전사 분석을 위해 RNA를 준비해야한다. 이 RNA의 인구는 수확 후 변경되지 않도록 꽃과 즉시 고정의 수확을 시작합니다. 조직은 임베디드 구분, 슬라이드에 장착된다. 이 LAM에 의해 세포 하나 또는 ?...

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

개별 세포 유형의 전사 프로파일 링을위한 도구로 레이저를 이용한 이외에 Microdissection (LAM)

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 ...

laser-assisted-microdissection-lam-as-tool-for-transcriptional

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

9.2K 조회수.  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. 

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

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

포유 동물 조직의 레이저 캡처 Microdissection

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

연구

생물학

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.

단일 셀 Transcriptome 분석

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

신경과학

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.

레이저 캡처 미세 해부 및 미세 유체 qPCR을 결합하여 단일 세포의 전사 프로필을 분석: 오피오이드 의존에 대한 시스템 생물학 접근법

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

유전학

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.

외상성 뇌 손상 후 뉴런 또는 유전자 발현 분석을위한 단일 뉴런의 강화 인구의 레이저 캡처 Microdissection

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.

유전자 발현 분석을 위한 마우스 배아 연골 및 뼈의 레이저 포획 미세 해부

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

발생학

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.

레이저 Microdissection는 세포의 하위 집합 유전자 발현 프로 파일링에 적용 Drosophila 윙 디스크

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

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.

레이저 캡처 이외에 Microdissection - 개별 도파민 신경 세포의 분리 및 전체 복부 피개 영역의 데모

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.

RNA 분석을위한 인간의 전립선 상피 세포의 레이저 캡처 이외에 Microdissection

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

의학

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.

셀 특정 부분 집단에서 RNA 분리 빠른 Immunolabeling과 결합 레이저 캡처 이외에 Microdissection을 사용하여

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.

종에 구애받지 않는 단일 조직 응용 분야를 위한 레이저 미세 해부

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