Nous remercions Molecular Devices technologies analytiques / Arcturus pour leur généreux don de réactifs utilisés pour l&#39;étude. Nous remercions également le Centre de Harvard ressources pour fournir des tissus post-mortem du tissu cérébral humain Financement:. RO1MH76060 et 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>

La production de cette vidéo article a été parrainé par Life Technologies.

Ici, nous essayons de personnaliser un protocole pour extraire les populations de cellules homogènes à partir de tissus post-mortem du cerveau hétérogènes utilisant le système d&#39;Arcturus XT et des kits d&#39;extraction d&#39;ARN liées. Comme indiqué plus haut, nous avons apporté quelques modifications aux protocoles originaux fournis par l&#39;entreprise, afin de maximiser l&#39;intégrité ARN. La capture réussie de cellules individuelles dépend uniquement de l&#39;optimisation des étapes avant la capture afin d&#39;atteindre les cellules au maximum avec de l&#39;ARN bon. Ces étapes comprennent les différents paramètres associés à des tissus de sectionnement et coloration. En bref, en ce qui concerne la préparation des tissus, notamment: 1) réduire le gradient de température lors de sectionnement, 2) que de placer deux sections par diapositive, 3) exécuter toutes les étapes de la déshydratation sur la glace, 4) en ajoutant l&#39;inhibiteur de RNase à la tache, 5) l&#39;augmentation la durée de déshydratation dans la finale d&#39;éthanol à 100% à 3 minutes. En ce qui concerne la capture, nous pensons que la combinaison de CapSure HS casquettes, avec le logiciel sur le réglage macro, devrait garantir des résultats optimaux. Un environnement à humidité contrôlée est critique pour la capture des cellules individuelles, que l&#39;humidité élevée réduit considérablement les tissus &quot;ascenseur&quot;. Des températures élevées peuvent aussi avoir un effet négatif sur la qualité de l&#39;ARN. Pendant le protocole d&#39;isolement d&#39;ARN avec le kit PicoPure, il ya deux étapes de lavage. Il est important de vérifier que tous les tampons de lavage est retirée avant le transfert à un autre tube à centrifuger pour l&#39;élution d&#39;ARN, comme toute présence de la mémoire tampon dans votre échantillon final se traduira par moins d&#39;ARN de départ pour l&#39;amplification. Pour ce faire, nous avons l&#39;étape de centrifugation lavage final pendant 2,5 minutes au lieu de 2 minutes comme le suggère le protocole fourni. Pour la plupart, le protocole d&#39;amplification linéaire a été conformément aux instructions du fabricant. Toutefois, deux points cruciaux doivent être pris en compte lors de l&#39;utilisation de ce protocole: 1) essayer de limiter la perte de l&#39;ARN par l&#39;intermédiaire de transférer l&#39;excès d&#39;échantillons entre les tubes, en utilisant uniquement des tubes 0.5ml fournis en conjonction avec un bloc de 0,5 ml dans le thermocycleur et 2) lors du transfert de l&#39;échantillon à la colonne de purification pour la purification de l&#39;ADNc, n&#39;oubliez pas de spin-down des tubes d&#39;échantillon après le premier transfert. Cela se traduira par un supplément de 1-2UL de l&#39;échantillon à ajouter à la colonne de purification et ne peut donc augmenter la récupération d&#39;ADNc. Lors de l&#39;utilisation du TURBO fin kit de marquage pour la biotine-étiquetage de vos échantillons ARNa limité, nous vous suggérons d&#39;ajouter un supplément de quelques ul d&#39;eau avant l&#39;étape de centrifugation, d&#39;augmenter le rendement de l&#39;ARN étiquetés extraite de la colonne. Une minute supplémentaire de centrifugation seront également utiles à cet égard, surtout si vous travaillez avec des tissus dégradés, tels que les tissus FFPE. Nous sommes convaincus que ce protocole permettra à l&#39;utilisateur d&#39;isoler de petites structures homogènes, comme les populations seule cellule, au sein de tissus post-mortem du cerveau hétérogène. Cela réduit les effets de dilution des structures environnantes et d&#39;autres types de cellules situées dans les différentes couches dans le cerveau, conduisant à des résultats ciblés vers les types de cellules spécifiques de l&#39;enquête. Comme la qualité de l&#39;ARN résultant est assez bon pour les études de puces à ADN, nous pouvons commencer à identifier des signatures moléculaires spécifiques des populations de cellules spécifiques affectés dans les états pathologiques.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>4.5&#x201D; Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-392</td>
<td>&nbsp;</td>
<tr>
<td>Arcturus XT&trade; Laser-Capture Microdissection (LCM) System</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>13821-00</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&trade; HS LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0214</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&reg; Macro LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0211</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; Automated Electrophoresis Station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7001</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; RNA HighSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7155</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA HighSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7156</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA StdSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7153</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&trade; RNA StdSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7154</td>
<td>10 chips</td>
</tr>
<tr>
<td>Falcon&reg; polypropylene Conical tube</td>
<td>&nbsp;</td>
<td>Becton-Dickinson Labware</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>GeneAmp&reg; thin-walled reaction tube with domed cap</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8010611</td>
<td>&nbsp;</td>
<tr>
<td>GeneChip&reg; Human X3P array</td>
<td>&nbsp;</td>
<td>Affymetrix</td>
<td>900516</td>
<td>6 chips</td>
</tr>
<tr>
<td>Histogene&reg; LCM Frozen Section Staining kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0401</td>
<td>For staining solution, jars, ethanols and Xylene</td>
</tr>
<tr>
<td>Histogene&trade; LCM slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>12231-00</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slide Box</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48444-004</td>
<td>&nbsp;</td>
<tr>
<td>Microm HM 505E cryostat</td>
<td>&nbsp;</td>
<td>Thermo Scientific</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>&nbsp;</td>
<td>Thermo Electron Corporation</td>
<td>51221048</td>
<td>Model 282</td>
</tr>
<tr>
<td>Molecular Sieve</td>
<td>&nbsp;</td>
<td>EMD&trade;</td>
<td>MX1583L-1</td>
<td>&nbsp;</td>
<tr>
<td>NanoDrop 1000 Spectrophotometer</td>
<td>&nbsp;</td>
<td>Thermo Scientific</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>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9932</td>
<td>&nbsp;</td>
<tr>
<td>PEN Membrane glass slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0522</td>
<td>&nbsp;</td>
<tr>
<td>PicoPure&reg; RNA Isolation kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0204</td>
<td>&nbsp;</td>
<tr>
<td>RiboAmp&reg;HSPLUS with Biotin labeling</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0511B</td>
<td>12 reactions Includes TURBO biotin labeling&trade; kit</td>
</tr>
<tr>
<td>RNase Zap&reg;</td>
<td>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9780/2</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>RNasin&reg; Plus</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N261B</td>
<td>&nbsp;</td>
<tr>
<td>SuperScript&trade; III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18080-044</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

Nous avons proposé d&#39;étudier la réduction de matière grise dans le gyrus temporal supérieur chez les patients schizophrènes, en interrogeant les profils d&#39;expression génique des neurones pyramidaux dans la couche III. Il est bien connu que le cortex cérébral est une structure extrêmement hétérogène comprenant diverses régions, des couches et des types de cellules, dont chacune est caractérisée par des compositions distinctes cellulaire et moléculaire et donc différentielle des profils d&#39;expression génique. Afin de contourner les effets confondants de l&#39;hétérogénéité des tissus, nous avons utilisé la capture microdissection laser (LCM) afin d&#39;isoler notre spécifiques de type cellulaire des neurones pyramidaux savoir. Environ 500 neurones pyramidaux colorés avec la solution de coloration ont été capturés à l&#39;aide Histogene l&#39;Arcturus LCM XT système. L&#39;ARN a ensuite été isolé à partir des cellules capturées et subi deux séries de T7 à base d&#39;amplification linéaire à l&#39;aide d&#39;Arcturus / Molecular Devices trousses. Le LabChip Experion (Bio-Rad) de gel et de bonne qualité électrophérogramme indique un ARN (m), avec une longueur de transcription étendre 600nt dernières requis pour biopuces. La quantité d&#39;ARNm obtenu en moyenne 51μg, avec la pureté des échantillons acceptables dire, comme indiqué par le ratio A260/280, de 2,5. L&#39;expression des gènes a été profilé à l&#39;aide du tableau de l&#39;homme X3P sonde GeneChip d&#39;Affymetrix.

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

Nous décrivons un processus utilisant le laser capture microdissection d&#39;isoler et d&#39;extraire l&#39;ARN à partir d&#39;une population cellulaire homogène, les neurones pyramidaux, dans la couche III du gyrus temporal supérieur dans les cerveaux post-mortem humain. Nous avons ensuite linéairement amplifier (T7-based) ARNm, et s&#39;hybrident l&#39;échantillon à la puce Affymetrix humaines X3P.

Obtention d&#39;ARN de haute qualité provenant des populations cellule unique de tissus post-mortem du cerveau de l&#39;homme

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

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30.5K vues.  McLean Hospital. Nous décrivons un processus utilisant le laser capture microdissection d'isoler et d'extraire l'ARN à partir d'une population cellulaire homogène, les neurones pyramidaux, dans la couche III du gyrus temporal supérieur dans les cerveaux post-mortem humain. Nous avons ensuite linéairement amplifier (T7-based) ARNm, et s'hybrident l'échantillon à la puce Affymetrix humaines X3P.

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

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Obtention des oeufs de Xenopus laevis femelles

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Recherche

Biologie

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.

Isolement des noyaux neuronaux à partir de tissus post-mortem du cerveau de l&#39;homme

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

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Génération d&#39;une seule cellule suspensions de tissus de souris de neurones

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Génération robuste des hépatocytes comme des cellules embryonnaires humaines à partir populations de cellules souches

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Génération de haute qualité Immunoprécipitation de la chromatine Patron de l&#39;ADN pour le séquençage à haut débit (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Un protocole de microdissection de capture de laser qui donne l'ARN de haute qualité des os frais-congelés de souris

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

Flux de travail protéomique quantitative utilisant la détection basée sur la surveillance des réactions multiples des protéines du tissu cérébral humain

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Prélèvement de tissus et extraction d’ARN de l’articulation ostéoarthritique humaine du genou

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.