Biz minnetle cömert bağışı için çalışma için kullanılan reaktiflerin Moleküler Cihazlar Analitik teknolojileri / Arcturus kabul etmiş oluyorsunuz. Ayrıca Harvard Doku Kaynağı Merkezi postmortem insan beyin dokusu sağlamak için teşekkür Finansman: RO1MH76060 ve P50MH080272.

<ol>
	<li>Imbeaud, S., Graudens, E., Boulanger, V., Barlet, X., Zaborski, P., Eveno, E., Mueleer, O., Schroeder, A., Auffray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Res. 33, e56-e56 (2005).</li></ol>

Bu video-makale üretim Yaşam Teknolojileri tarafından sponsor oldu.

Burada, Arcturus XT sistemi ve ilgili RNA ekstraksiyon kitleri kullanarak heterojen postmortem beyin dokusu homojen hücre popülasyonlarının ayıklamak için bir protokol özelleştirmek için çalışırlar. Yukarıda belirtildiği gibi, RNA bütünlüğünü maksimize etmek için, şirket tarafından sağlanan orijinal protokollere bazı değişiklikler yaptık. Tek hücre başarılı yakalama sadece iyi RNA ile maksimum hücreleri elde etmek için yakalamak için önceki adımları optimize bağlıdır. Bu adımlar, doku kesit ve boyama ile ilgili çeşitli parametreler içerir. Kısacası, bu doku hazırlanması konusunda şunlardır: 1), 2 kesit sıcaklık gradyanı azalan) sadece 4 dehidrasyon, buz üzerinde tüm adımları gerçekleştirdikten slayt başına 2 bölüm, 3) yerleştirerek) artan) Leke, 5 RNaz İnhibitörü ekleyerek 3 dakika için son% 100 etanol dehidrasyon süresi. Ilgili yakalama, biz CapSure HS kapaklar, Makro ayarı yazılımı ile kombinasyonu optimum sonuçlar sağlamak gerektiğini hissediyorum. Nem kontrollü bir ortamda, yüksek nem, doku &quot;asansör&quot; önemli ölçüde azaltır olarak yakalayan tek hücre için çok önemlidir. Yüksek sıcaklıklar da RNA kalitesi üzerinde olumsuz bir etkisi olabilir. PicoPure kiti ile RNA izolasyonu protokol sırasında, iki yıkama adımlar vardır. Bu RNA yürütülmesi için başka bir mikrosantrifüj tüp aktarmadan önce, tüm yıkama tamponu kaldırıldı olup olmadığını kontrol edin, son örnek tampon herhangi bir varlığı olarak amplifikasyonu için daha az başlangıç ​​RNA neden olacaktır önemlidir. Bunu sağlamak için, verilen protokol önerilen 2.5 dakika yerine 2 dakika için son yıkama adım santrifüj. Doğrusal amplifikasyon protokolü büyük bir kısmı için, üreticinin talimatlarına göre. Ancak, bu protokolü kullanırken iki önemli noktayı dikkate alınması gerekmektedir: 1) borular arasında, sadece termal cycler 0.5 ml bloğu ile birlikte sağlanan 0.5 ml tüpler kullanarak aşırı örneklerin aktarılması yoluyla RNA kaybının deneyin ve 2) için örnek cDNA arıtılması için arıtma sütununa aktarırken, ilk transferinden sonra örnek tüpler aşağı spin emin olun. Bu arıtma sütun eklemek ve bu nedenle cDNA kurtarma artırabilir örnek bir ek 1 2ul neden olacaktır. TURBO son etiketleme kiti kullanımı sınırlı Arna örnekleri biotin-etiketlenmesi için yaparken, ekstra bir kaç ul ekleyerek etiketli RNA sütun çıkarılan verimi artırmak, santrifüj adım önce su öneririz. Santrifüj ekstra bir dakika da FFPE doku gibi bozulmuş doku ile çalışıyoruz, özellikle eğer bu konuda yardımcı olacaktır. Biz bu protokolü, heterojen postmortem beyin dokusu içinde tek bir hücre nüfus, küçük homojen yapılar, izole etmek için kullanıcı sağlayacaktır emin hissediyorum. Bu soruşturma altında özel hücre tipleri doğru hedeflenen sonuçlara yol açan beyin içinde çeşitli katmanlarında bulunan çevreleyen yapılar ve diğer hücre türleri sulandırma etkileri azaltır. Ortaya çıkan RNA kalitesini mikroarray çalışmaları için yeterli olduğu gibi, belirli bir hücre popülasyonlarının hastalık durumlarında etkilenen spesifik moleküler imzaları saptamak için başlayabilir.

<table><tbody><tr><td>4.5&amp;#8221; Forceps</td><td>VWR international</td><td>82027-392</td><td></td></tr><tr><td>Arcturus XT&amp;#8482; Laser-Capture Microdissection (LCM) System</td><td>MDS Analytical Technologies</td><td>13821-00</td><td></td></tr><tr><td>CapSure&amp;#8482; HS LCM Caps</td><td>MDS Analytical Technologies</td><td>LCM 0214</td><td></td></tr><tr><td>CapSure&amp;#174; Macro LCM Caps</td><td>MDS Analytical Technologies</td><td>LCM 0211</td><td></td></tr><tr><td>Experion&amp;#8482; Automated Electrophoresis Station</td><td>Bio-Rad</td><td>700-7001</td><td></td></tr><tr><td>Experion&amp;#8482; RNA HighSens Chips</td><td>Bio-Rad</td><td>700-7155</td><td>10 chips</td></tr><tr><td>Experion&amp;#8482; RNA HighSens Reagents</td><td>Bio-Rad</td><td>700-7156</td><td>10 chips</td></tr><tr><td>Experion&amp;#8482; RNA StdSens Chips</td><td>Bio-Rad</td><td>700-7153</td><td>10 chips</td></tr><tr><td>Experion&amp;#8482; RNA StdSens Reagents</td><td>Bio-Rad</td><td>700-7154</td><td>10 chips</td></tr><tr><td>Falcon&amp;#174; polypropylene Conical tube</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>GeneAmp&amp;#174; thin-walled reaction tube with domed cap</td><td>Applied Biosystems</td><td>N8010611</td><td></td></tr><tr><td>GeneChip&amp;#174; Human X3P array</td><td>Affymetrix</td><td>900516</td><td>6 chips</td></tr><tr><td>Histogene&amp;#174; LCM Frozen Section Staining kit</td><td>MDS Analytical Technologies</td><td>KIT0401</td><td>For staining solution, jars, ethanols and Xylene</td></tr><tr><td>Histogene&amp;#8482; LCM slides</td><td>MDS Analytical Technologies</td><td>12231-00</td><td></td></tr><tr><td>Micro Slide Box</td><td>VWR international</td><td>48444-004</td><td></td></tr><tr><td>Microm HM 505E cryostat</td><td>Thermo Fisher Scientific, Inc.</td><td>22-050-350</td><td>Catalogue number for similar product as current product no longer available</td></tr><tr><td>Microprocessor Controlled 280 series Water bath</td><td>Thermo Fisher Scientific, Inc.</td><td>51221048</td><td>Model 282</td></tr><tr><td>Molecular Sieve</td><td>EMD Millipore</td><td>MX1583L-1</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td>n/a</td><td>The NanoDrop 1000 is not available anymore, but the NanoDrop 2000 is comparable.</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9932</td><td></td></tr><tr><td>PEN Membrane glass slides</td><td>MDS Analytical Technologies</td><td>LCM 0522</td><td></td></tr><tr><td>PicoPure&amp;#174; RNA Isolation kit</td><td>MDS Analytical Technologies</td><td>KIT0204</td><td></td></tr><tr><td>RiboAmp&amp;#174;HS&lt;sup&gt;PLUS&lt;/sup&gt; with Biotin labeling</td><td>MDS Analytical Technologies</td><td>KIT0511B</td><td>12 reactions Includes TURBO biotin labeling&amp;#8482; kit</td></tr><tr><td>RNase Zap&amp;#174;</td><td>Ambion</td><td>AM9780/2</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>RNasin&amp;#174; Plus</td><td>Promega Corp.</td><td>N261B</td><td></td></tr><tr><td>SuperScript&amp;#8482; III Reverse Transcriptase</td><td>Invitrogen</td><td>18080-044</td><td></td></tr></tbody></table>

obtaining high quality rna from single cell populations in human postmortem brain tissue

Biz tabaka III piramidal nöronların gen ekspresyon profilleri sorgulayarak, şizofreni hastalarında görülen superior temporal girus gri cevherde azalma araştırmak için önerdi. Serebral korteks, farklı bölgelerde, katmanlar ve hücre tipleri, her biri farklı hücresel ve moleküler kompozisyon ve bu nedenle diferansiyel gen ekspresyon profilleri ile karakterize oluşan son derece heterojen bir yapısı olduğu bilinmektedir. Doku heterojenliğin karıştırıcı etkilerini aşmak için, belirli bir hücre tipi yani piramidal nöronlar izole etmek için lazer yakalama mikrodiseksiyon (LCM) kullanılır. Histogene boyama solüsyonu ile boyanmış yaklaşık 500 piramidal nöronlar Arcturus XT LCM sistemini kullanarak ele geçirildi. RNA daha sonra yakalanan hücreleri izole ve iki tur T7 tabanlı doğrusal amplifikasyon Arcturus / Moleküler Cihazlar kitleri kullanılarak uygulandı. Experion LabChip (Bio-Rad) jel ve Elektroferogram mikroarray&#39;ler için gerekli geçmiş 600nt uzanan bir transkript uzunluğu ile, kaliteli bir (m) RNA belirtilir. 2.5 A260/280 oranı, belirtildiği gibi, elde edilen mRNA miktarı kabul edilebilir ortalama örnek saflıkta, 51μg ortalama. Gen ekspresyonu Affymetrix İnsan X3P GeneChip prob dizi ile profilli oldu.

Watch this Scientific Journal Video about Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue at JoVE.com

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

Biz postmortem insan beyni superior temporal girus III. Tabakası homojen bir hücre popülasyonu, piramidal nöronlar, RNA izole etmek ve ayıklamak için lazer yakalama mikrodiseksiyon kullanarak bir süreci tanımlar. Biz (T7 tabanlı) mRNA daha sonra doğrusal yükseltmek. Kullanmakta olduğunuz örnek Affymetrix insan X3P mikroarray melezleşme.

İnsan Postmortem Beyin Dokusu Tek Hücre Popülasyonlar Yüksek Kalite RNA Alınması

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

obtaining-high-quality-rna-from-single-cell-populations-human

Research

JoVE Journal

Biology

30.6K Görüntüleme.  McLean Hospital. Biz postmortem insan beyni superior temporal girus III. Tabakası homojen bir hücre popülasyonu, piramidal nöronlar, RNA izole etmek ve ayıklamak için lazer yakalama mikrodiseksiyon kullanarak bir süreci tanımlar. Biz (T7 tabanlı) mRNA daha sonra doğrusal yükseltmek. Kullanmakta olduğunuz örnek Affymetrix insan X3P mikroarray melezleşme.

Video: Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

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.

Tek Hücre Transcriptome Analizi

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

Nörobilim

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.

Toplam RNA İzolasyonu ve Alt Nesil Sıralama ve Kaynak gösterilmeden kullanılamaz Ayrık Fare Beyin Bölgeler Mikrodisseksiyon Sigara Lazer Yakalama Mikroskopi Yaklaşım

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

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

İnsan Postmortem Beyin Dokusu Nöronal Çekirdekler İzolasyon

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Biyoloji

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Tek Çekirdekli RNA-seq ve ATAC-seq için Taze Donmuş Beyin Tümörlerinden Çekirdek İzolasyonu

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

Kanser Araştırmaları

İnsan Beyni Organoidlerinden Tek Hücre ve Çekirdek Ekstraksiyonu

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

Yazar Spotlight: Organoid Modellerin Tek Hücreli ve Uzamsal Transkriptomik Teknolojilerle Entegre Edilmesi

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

Beyin Organoidlerinde Tek Hücreli ve Tek Çekirdekli Transkriptomların Oluşumu ve Aşağı Akış Analizi

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

Gelişim Biyolojisi

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Yetişkin omurilik çekirdeği yalıtım kitlesel paralel tek-çekirdek RNA sıralama için

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Yüksek kaliteli lazer RNA izolasyonu için paslanmaz bir protokol yakalama insan öldükten sonra beyincik Microdissected Purkinje hücreleri

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

İnsan Postmortem Beyin Dokusu Tek Hücre Popülasyonlar Yüksek Kalite RNA Alınması

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Tek Hücre Transcriptome Analizi

Toplam RNA İzolasyonu ve Alt Nesil Sıralama ve Kaynak gösterilmeden kullanılamaz Ayrık Fare Beyin Bölgeler Mikrodisseksiyon Sigara Lazer Yakalama Mikroskopi Yaklaşım

İnsan Postmortem Beyin Dokusu Nöronal Çekirdekler İzolasyon

Tek Çekirdekli RNA-seq ve ATAC-seq için Taze Donmuş Beyin Tümörlerinden Çekirdek İzolasyonu

Beyin Organoidlerinde Tek Hücreli ve Tek Çekirdekli Transkriptomların Oluşumu ve Aşağı Akış Analizi

Beyin Organoidlerinde Tek Hücreli ve Tek Çekirdekli Transkriptomların Oluşumu ve Aşağı Akış Analizi

Beyin Organoidlerinde Tek Hücreli ve Tek Çekirdekli Transkriptomların Oluşumu ve Aşağı Akış Analizi

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