우리는 기증자와이 일을 가능 하 게 그들의 가족에 감사입니다. 우리는 또한 국제 의학 연구소 및 테네시 기증자 서비스 조직의이 연구에 사용 된 좌표 컬렉션을 돕는 직원의 도움을 주셔서 감사 합니다. 이 작품은 건강의 국가 학회, NIH OT2-OD023850 EMS2 와 봉급 지원 NIH에서 T32 일부-DK007673 AMZ 하에서 교부 금에 의해 지원 되었다. 우리는 밴 더 빌 트 변환 병리학 공유 리소스의 직원 조직 준비에 조언과 LCM 악기에 대 한 액세스 감사입니다. 밴 더 빌 트 조직 병리학 공유 리소스 NIH P30-CA068485-14 보조금과 U24-DK059637-13에 의해 부분에서 지원 됩니다. 우리는 게놈 분석에 대 한 게놈 기술 접근 센터 워싱톤 대학의과 대학에 유전학의 부에 감사합니다. GTAC는 부분적으로 지원 됩니다 Siteman 암 센터 NCI 암 센터 지원 그랜트 #P30 CA91842와 국립 센터에서 ICTS/CTSA 그랜트 #UL1TR002345 연구 자원 (NCRR), 국립 보건원 (NIH), 그리고 NIH의 구성 요소에 대 한 의료 연구에 대 한 로드맵입니다. 이 간행물은 전적으로 저자의 책임 고 반드시 NCRR 또는 NIH의 공식 보기를 대표 하지 않는다.

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저자는 공개 관심의 없습니다 충돌 있다.

이 절차 RNA-이 대 한 RNA를 파생 하는 원본으로 수많은 장 중추의 효율적인 수집 가능 여기, 우리가 극대로 RNA 무결성을 유지 하면서 기존 프로토콜에서 설명 하는 프로세스를 가속 있다. 이 절차의 모든 단계는 상호 의존, 그것은 중요 한 연구의 개시에서 모든 문제를 제거 하 고는 모든 샘플 준비가 되어로 유사 하 게 다른 가능한 신뢰할 수 있는 RNA-이를
절차의 개시에서 RNA 무...

이 메서드는 레이저 캡처 기정 (LCM)를 사용 하 여 인간의 창 자 조직에서 장의 중추의 높은-품질 RNA 샘플을 얻기 위해 설계 되었습니다. 여기에 설명 된 프로토콜 RNA 시퀀싱 (RNA-seq)에 대 한 충분 한 RNA 품질 및 수율을 제공 하도록 최적화 된 하며 갓 절제, 고정, 플래시-냉동 인간 창 자 조직으로 사용 될 것입니다.
기능성 위장 하 고 본질적인 운동 성 장애는 미국에서 4 명 중 하나에 영향을 줍니다. 장 신 경계 (ENS), 두 번째 뇌1라고도 종종 이러한 장애의 중심에 직감 항상성 및 운동에 중요 한 역할. 창 자 운동 성의 조작 일반적으로 aganglionic/noncontractile 조직, 만성 식이 수정 및/또는 약물의 외과 절제술으로 제한 되었습니다. 놀랍게도, 성인 팔의 전체 transcriptome 시퀀싱 할 약물 타겟 줄기 세포 치료에 활용 될 수 있는 ENS 내의 분자를 식별 하는 우리의 기능을 크게 제한 남아 있습니다.
인간의 장 중추에서 RNA를 분리 하는 데 상대적으로 몇 가지 방법이 있다. 첫 번째 접근 방법, 셀 분리2, 필요 높은 부 화 온도 및 긴 보육 시간; 모두는 RNA 저하를 촉진 하 고 변경 transcriptome2,3으로 알려져 있습니다. 다른 접근 방법, LCM, 더 안정적으로 transcriptome를 유지 하 고 RNA 무결성 보호. 이러한 방법 중 가난한 RNA 품질 및 수량에 의해 방해 했다, 아주 노동 집약, 여러 연구는 사용 하지만 LCM 신선한-냉동 인간 창 자 조직4,,56에서 중추를 수집, 또는 얼룩 또는 RNA 추출 기술을 우리의 손에서 일의 필요한 수정. LCM 제품 설명서 제공 장의 중추8, 의 분리에 적용 될 때 추가 개선7,8, 하지만 적응 필요에서 발견 된 다른 LCM 프로토콜 RNA를 보존 하기 위한 9. 이러한 이유로 우리는 최적화 된 프로토콜을 기반으로 이러한 리소스에는 인간의 장 중추에서 상당한 양의 높은 무결성 RNA 생성, 상대적으로 빠른 워크플로우, 그리고 대형에 걸쳐 일관 된 결과 생성을 개발 샘플의 수입니다.
이 연구에서 우리는 절제 인간의 창 자 조직에서 공급 하는 장 신경에서 높은 무결성 RNA의 분리를 용이 하 게 하는 최적화 된 절차의 개요를 제시. 우리의 방법 5 가지 중요 한 측면을 통합합니다. 첫째, 갓 절제, 고정 되지 않은 인간의 장 샘플 크기, 모든 과잉 습기 실험실 티슈로 제거 하 고 2-methylbutane (2 MB)과 드라이 아이스의 슬러리 꼭대기 플래시-냉동 하기 전에 큰 기본 몰드에 평평 하 게 손질 한다. 내장의 두 번째, 더 섹션 장의 중추의 큰 페이로드를 제공 하는 슬라이드에 myenteric 총의 전체 평면을 얻기 위해 준비 되어야 한다. 성공이 단계는 조직 준비 과정에 크게 의존 합니다. 셋째, 중추는 존재에의 비균일 구조 폴 리 에틸렌 napthalate (펜) 막 슬라이드6, LCM 과정에서 최고의 속도 정밀도 제공 하는 사용을 해야 합니다. Cresyl 바이올렛 등 4, 에탄올 호환 염료 장의 중추 얼룩 동안 RNA 무결성을 유지 하기 위해 사용 되어야 한다. 마지막으로, RNA 추출 과정은 RNA-시퀀스와 성공적인 결과 위해 중요 한 우리는 높은 RNA 무결성, 장 신경의 작은 컬렉션을 시작할 때 RNA 수율을 극대화, DNA 오염 제거 하 고 가능한 많은 RNA 종 유지 RNA 추출 방법을 모색.
함께 찍은, 현재 연구에서 이러한 요소의 최적화 크게 가속 워크플로 및 장의 중추 뛰어난 RNA 수량 및 품질의 샘플을 생성. 결과이 접근의 일관성을 나타내는 샘플의 상당한 그룹 간에 크게 일관 되었습니다. 또한, 이러한 접근 수십 장의 중추에서 RNA 샘플을 성공적으로 시퀀싱을 사용 했습니다. 여기에서 강조 하는 전략 수행 원하는 중추의 LCM 또는 주변 장치 및 중앙 신 경계와 높은-품질 RNA의 절연이 필요한 다른 경우의 핵에 대 한 광범위 하 게 적응 수 있습니다 또한.

<table><tbody><tr><td>10% Neutral-buffered formalin</td><td>Sigma</td><td>HT501128-4L</td><td></td></tr><tr><td>2-Methylbutane</td><td>Fisher</td><td>O3551-4</td><td></td></tr><tr><td>3-mL syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>50-mL conical tubes, polypropylene&amp;nbsp;</td><td>Corning</td><td>05-526B</td><td></td></tr><tr><td>Base molds 37x24x5mm, disposable</td><td>Electron Microscopy Sciences</td><td>5025511</td><td></td></tr><tr><td>Belzer UW&amp;nbsp; Cold Storage Solution</td><td>Bridge to Life</td><td>N.A.</td><td></td></tr><tr><td>CapSure Macro LCM caps</td><td>Arcturus, ThermoFisher</td><td>LCM0211</td><td></td></tr><tr><td>Cling Wrap, Press&amp;rsquo;n Seal&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;Glad&amp;nbsp;&amp;nbsp;</td><td>N.A.&amp;nbsp;</td><td></td></tr><tr><td>Cresyl Violet Acetate</td><td>Acros Organics</td><td>AC405760025</td><td></td></tr><tr><td>Cutting Board</td><td>Electron Microscopy Sciences</td><td>63308</td><td></td></tr><tr><td>Ethanol, 190-proof</td><td>Pharmco-AAPER</td><td>111000190</td><td></td></tr><tr><td>Ethanol, 200-proof</td><td>Pharmco-AAPER</td><td>111000200</td><td></td></tr><tr><td>Glass Microscope slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Gloves, extended cuff</td><td>Microflex&amp;nbsp;</td><td>19010144</td><td></td></tr><tr><td>Gowns, surgical-disposable</td><td>Kimberly-Clark&amp;nbsp;</td><td>19-088-2116</td><td></td></tr><tr><td>Tray, 20 L (large polypropylene sterilizing pan)</td><td>Nalgene&amp;nbsp;&amp;nbsp;</td><td>1335920B</td><td></td></tr><tr><td>Kimwipes (large)</td><td>Kimberly-Clark&amp;nbsp;</td><td>34120</td><td></td></tr><tr><td>Kimwipes (small)</td><td>Kimberly-Clark&amp;nbsp;</td><td>34133</td><td></td></tr><tr><td>Laser Capture Microdissection System (ArcturusXT)</td><td>ThermoFisher</td><td>N.A.</td><td></td></tr><tr><td>Leica Cryostat Chuck, large,&amp;nbsp; 40 mm&amp;nbsp;</td><td>Southeast Pathology Instrument Service</td><td>N.A.&amp;nbsp;</td><td></td></tr><tr><td>Light Microscope</td><td>Olympus</td><td>CX43</td><td></td></tr><tr><td>Microscope objective (20X)</td><td>Olympus UPlanFl 20X/0.50, &amp;infin;/0.17</td><td>N.A.</td><td></td></tr><tr><td>Microscope objective (10X)</td><td>Olympus UPlanFl 10X/0.25, &amp;infin;/-</td><td>N.A.&amp;nbsp;</td><td></td></tr><tr><td>Molecular Sieves</td><td>Acros Organics</td><td>AC197255000</td><td></td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>PicoPure RNA extraction kit</td><td>Applied Biosystems</td><td>12204-01</td><td></td></tr><tr><td>Pellet Pestle</td><td>Kimble Kontes</td><td>4621973</td><td></td></tr><tr><td>PEN membrane LCM slides</td><td>Arcturus, ThermoFisher</td><td>LCM022</td><td></td></tr><tr><td>RNase-free tubes, 1.5 mL</td><td>Ambion</td><td>AM12400</td><td></td></tr><tr><td>RNaseZAP&amp;nbsp;</td><td>Sigma</td><td>Sigma-R2020</td><td></td></tr><tr><td>RNA Extraction Kit &quot;A&quot; (PicoPure)</td><td>Applied Biosystems</td><td>12204-01</td><td></td></tr><tr><td>RNA Extraction Kit &quot;B&quot; (RNeasy&amp;nbsp; Micro kit)</td><td>Qiagen</td><td>74004</td><td></td></tr><tr><td>RNA Extraction Method &quot;C&quot; (TRIzol)</td><td>Invitrogen</td><td>15596-026</td><td></td></tr><tr><td>RNA Extraction Kit &quot;D&quot; (RNeasy PLUS Mini kit)</td><td>Qiagen</td><td>74134</td><td></td></tr><tr><td>Splash Shield, disposable faceshield</td><td>Fisher</td><td>17-310</td><td></td></tr><tr><td>Scissors, Surgical, 14.5 cm &quot;sharp-sharp&quot;</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>Syringe filter, cellulose acetate (0.2 &amp;mu;m)</td><td>Nalgene&amp;nbsp;&amp;nbsp;</td><td>190-2520</td><td></td></tr><tr><td>Tissue Freezing Medium</td><td>General Data Healthcare</td><td>TFM-5</td><td></td></tr><tr><td>Xylenes</td><td>Fisher</td><td>X3P1GAL</td><td></td></tr></tbody></table>

optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue

이 방법의 목적은 고정, 갓 절제 인간의 창 자 조직 레이저 캡처 기정 (LCM)를 사용 하 여에서 수집 하는 장 신경에서 높은 무결성 RNA 샘플을 얻을 것입니다. 5 단계 워크플로에서 RNA-이 대 한 충분히 높은 품질과 수량 장의 중추에서 RNA 분리를 얻기위한 중요 한를 확인 했습니다. 첫째, 준비할 때 장 조직, 각 샘플 아래쪽 큰 기본 금형의 가능한 serosa 병합 전에 blotting에 의해 제거 하는 모든 초과 액체를가지고 있어야 합니다. 다음 샘플 2 methylbutane 드라이 아이스의 슬러리 꼭대기 신속 하 게 고정 됩니다. 둘째, 조직 단면, 그것 장 섹션 슬라이드 당 장 신경의 가장 큰 표면 영역을 양보 함으로써 myenteric 총의 전체 평면 병렬 cryomolds 위치에 중요 하다. LCM, 폴 리 에틸렌 napthalate (펜) 중 세 번째-최고의 속도 장의 중추를 수집 하는 때 장 중추의 비균일 도형의 윤곽에 유연성을 제공 하는 막 슬라이드. 넷째, 섹션 내 장 신경의 고유한 시각화를 위한 Cresyl 바이올렛 같은 에탄올 호환 염료 수성 염료를 기준으로 RNA 무결성의 우수한 보존을 제공합니다. 마지막으로, 캡처된 중추에서 RNA 추출, 우리는 DNA 오염을 제거 하는 동안 우수한 RNA 수량 나왔고 상업적인 RNA 추출 키트와 품질, 차이점 관찰. 현재 프로토콜에서 이러한 요소의 최적화 크게 워크플로우를 가속화 하 고 뛰어난 RNA 품질 및 수량 장의 중추 샘플을 생성 합니다.

기존 프로토콜 RNA-이 대 한 기준을 충족 하는 LCM을 사용 하 여 인간의 장 샘플에서 장의 중추의 상대적으로 빠른 컬렉션을 사용할 수 있는 여러 가지 개선을 하였습니다. 첫째, 우리는 2-mb (그림 1B) 드라이 아이스의 슬러리의 표면에 배치 하는 큰 기본 금형에서 장 조직 세그먼트의 급속 동결 최적화. Cryosectioning 및 후속 LCM 동안 최고의 성?...

Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

신선한 냉동 인간 조직에서 장의 중추의 격리에 대 한 레이저 캡처 서의 최적화

이 프로토콜의 목표에서 고정, 갓 절제 인간의 창 자 조직 레이저 캡처 기정 (LCM)를 사용 하 여 격리 장 중추에서 높은 무결성 RNA 샘플을 얻을 것입니다. 이 프로토콜 포함 플래시-냉동 인간 창 자 조직, cryosectioning, ethanolic 얼룩 및 탈수, LCM, 샘플 준비 및 RNA 추출.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

optimization-laser-capture-microdissection-for-isolation-enteric

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.6K 조회수.  Vanderbilt University Medical Center. 이 프로토콜의 목표에서 고정, 갓 절제 인간의 창 자 조직 레이저 캡처 기정 (LCM)를 사용 하 여 격리 장 중추에서 높은 무결성 RNA 샘플을 얻을 것입니다. 이 프로토콜 포함 플래시-냉동 인간 창 자 조직, cryosectioning, ethanolic 얼룩 및 탈수, LCM, 샘플 준비 및 RNA 추출.

비디오: Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

연구

신경과학

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.

총 RNA의 분리 및 다운 스트림 차세대 시퀀싱 및 유전자 발현 프로 파일링에 대한 개별 마우스 뇌 영역의 Microdissection 아닌 레이저 캡처 현미경 접근법

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

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

수술 표본에서 인간 췌장 섬의 레이저 Microdissection을위한 개선 된 프로토콜

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

의학

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 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 Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

종양 내 이질성, 온코스트림 및 침략의 공간 및 분자 특성화를 위한 신경교종 소구역의 레이저 캡처 미세 분해

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

단일 핵 RNA 시퀀싱을 위한 마우스 교감신경 신경절의 바코드된 항체를 사용한 저입력 핵 분리 및 멀티플렉싱

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

레이저 캡처 서 유전자 표정 분석에 대 한 마우스 눈에서 높은 순수 배수 채널의

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

생물학

Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model

Source: Bullock M. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/51475"> Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and&#160;Ex vivo&#160;Fibroblasts.</a> J. Vis. Exp. (2014).
The video describes the laser dissection of the 3D organotypic co-culture model with micron-level precision. In 3D organotypic models, stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells. The 3D model serves as an experimental tool that enables invasion and cancer-stroma interactions to be studied in near-physiological conditions.

레이저 미세해부: 3D 유기형 배양 모델에서 침입하는 대장암 세포를 분리하는 기술

Source: Bullock M. et al.,  Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and&#160;Ex ...

JoVE Encyclopedia of Experiments

암 연구학

Colorectal Cancer

Assays & techniques

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Submerge the slides in 30-second sequential descending ethanol immersions, followed by crystal violet solution staining for 20 seconds and 5 seconds in 0.5% eosin Y solution.
At the end of the eosin Y incubation, use filter paper to blot the slides dry and dehydrate the slides in sequential ascending ethanol immersions. After the 60-second ethanol immersion, rinse the slides in a container of xylene for 3 minutes.
Next, dry the slides on RNase-free surface at room temperature for 10 seconds before mounting the samples in mounting medium prepared with RNase-free water. After 10 to 20 seconds, transfer the slides onto the microscope microdissection platform.