Wir bedanken uns für Molecular Devices Analytical Technologien / Arcturus für die großzügige Spende von Reagenzien für die Studie verwendet. Wir danken auch der Harvard Tissue Resource Center für die Bereitstellung von postmortalen menschlichen Hirngewebe Finanzierung:. RO1MH76060 und 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>

Die Produktion dieses Video-Artikel wurde von Life Technologies gesponsert.

Hier versuchen wir, ein Protokoll anzufertigen, um homogene Zellpopulationen aus heterogenen postmortalen Hirngewebe mit dem Arcturus XT-System und verwandte RNA Extraktion Kits zu extrahieren. Wie oben dargelegt, haben wir einige Änderungen an dem ursprünglichen Protokolle, die von dem Unternehmen zur Verfügung gestellt hat, um RNA-Integrität zu maximieren. Die erfolgreiche Erfassung der einzelnen Zellen hängt einzig und allein auf die Optimierung der Schritte vor der Aufnahme um ein Höchstmaß an Zellen mit guter RNA zu erreichen. Diese Schritte umfassen die verschiedenen Parameter mit Gewebe Schneiden und Färben verbunden. Kurz gesagt, in Bezug auf Gewebe Vorbereitung dazu gehören: 1) Verringerung der Temperaturgradienten beim Schneiden, 2) nur Platzierung 2 Sektionen pro Objektträger, 3) die Durchführung aller Dehydration Schritte auf Eis, 4) Zugabe von RNase Inhibitor, um den Fleck, 5) erhöht die Dehydratisierung Dauer in den letzten 100% Ethanol bis 3 Minuten. Im Hinblick auf die Erfassung, fühlen wir, dass die Kombination von CapSure HS Kappen, mit der Software auf Makro-Einstellung, sollte für optimale Ergebnisse zu gewährleisten. Ein Feuchte-kontrollierten Umgebung ist entscheidend für die Erfassung von einzelnen Zellen, wie hohe Luftfeuchtigkeit drastisch reduziert Gewebe &quot;lift&quot;. Hohe Temperaturen können auch einen negativen Effekt auf die RNA-Qualität. Während der RNA-Isolierung Protokoll mit dem PicoPure Kit gibt es zwei Waschschritte. Es ist wichtig zu überprüfen, ob alle Waschpuffer vor der Übertragung auf ein anderes Mikrozentrifugenröhrchen für die RNA-Elution entfernt ist, wie jede Gegenwart des Puffers in die endgültige Probe wird in weniger Ausgangs-RNA zur Verstärkung führen. Um dies zu gewährleisten, Zentrifuge wir die letzte Waschschritt für 2,5 Minuten statt 2 Minuten, wie im Protokoll vorgesehen vorgeschlagen. Zum größten Teil wurde die lineare Verstärkung Protokoll gemäß den Anweisungen des Herstellers. Allerdings sollten zwei wichtige Punkte berücksichtigt werden, wenn dieses Protokoll verwenden: 1) versuchen, um den Verlust der RNA über mehr als die Übertragung von Proben Grenze zwischen Rohren, indem nur die 0,5 ml Röhrchen in Verbindung mit einem 0,5 ml-Block in den Thermocycler zur Verfügung gestellt und 2) bei der Übertragung der Probe auf die Reinigung Spalte für die Reinigung von cDNA, achten Sie darauf, Spin-down der Probenröhrchen nach der ersten Übertragung. Dies wird in einer zusätzlichen 1-2UL der Probe auf die Reinigung Spalte hinzuzufügen und kann daher cDNA Besserung steigt führen. Bei der Nutzung der TURBO End-Labeling Kit für Biotin-Markierung von Ihrer begrenzten aRNA Proben, empfehlen wir Ihnen das Hinzufügen eines zusätzlichen wenigen ul ist von Wasser vor dem Zentrifugationsschritt zur Erhöhung der markierten RNA aus der Säule entnommen Ertrag. Eine zusätzliche Minute Zentrifugation wird auch in dieser Hinsicht helfen, besonders wenn Sie mit dem abgebauten Gewebe, wie FFPE Gewebe arbeiten. Wir sind zuversichtlich, dass dieses Protokoll wird dem Benutzer zu ermöglichen kleinen homogenen Strukturen wie einzelne Zellpopulationen in heterogenen postmortalen Hirngewebe zu isolieren. Dies reduziert Verwässerungseffekte von umgebenden Strukturen und anderen Zelltypen in den verschiedenen Schichten innerhalb des Gehirns lokalisiert ist, was die Ergebnisse auf die spezifischen Zelltypen untersucht ausgerichtet. Da die Qualität der resultierenden RNA ist gut genug für Microarray-Studien, können wir beginnen, spezifische molekulare Signaturen von spezifischen Zellpopulationen in Krankheit betroffenen Staaten lokalisieren.

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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

Wir schlugen vor, die graue Substanz Reduzierung der oberen temporalen Gyrus bei Schizophrenie-Patienten gesehen zu untersuchen, durch Abfrage Genexpressionsprofile Pyramidenneuronen in Schicht III. Es ist bekannt, dass die Großhirnrinde einen außergewöhnlich heterogene Struktur aus unterschiedlichen Regionen, Schichten und Zelltypen, die jeweils durch verschiedene zelluläre und molekulare Zusammensetzung und somit differenziellen Genexpression Profile gekennzeichnet ist. Zur Umgehung der verwirrenden Wirkungen einer Gewebekultur Heterogenität, haben wir Laser-Mikrodissektion (LCM), um unsere spezifischen Zelltyp dh Pyramidenneuronen isolieren. Etwa 500 Pyramidenneuronen mit dem Histogene Färbelösung gefärbt wurden unter Verwendung des Arcturus XT LCM-System. RNA wurde dann von eingefangenen Zellen isoliert und unterzog sich zwei Runden T7-basierte lineare Verstärkung mit Arcturus / Molecular Devices Kits. Die Experion LabChip (Bio-Rad)-Gel und Elektropherogramm angegeben guter Qualität a (m) RNA, eine Abschrift Länge erstreckt Vergangenheit 600nt für Microarrays erforderlich. Die Menge der mRNA erreicht durchschnittlich 51μg, mit akzeptablen Mittelwert Probenreinheit wie von der A260/280 Ratio von 2,5 angegeben. Die Genexpression wurde profiliert mit dem Human X3P GeneChip Sondenarray von 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

Wir beschreiben ein Verfahren mittels Laser-Mikrodissektion zu isolieren und zu extrahieren RNA aus einer homogenen Zellpopulation, pyramidalen Neuronen in der Schicht III des oberen temporalen Gyrus in postmortalen menschlichen Gehirnen. Wir anschließend linear verstärken (T7-based) mRNA und hybridisieren die Probe auf die Affymetrix menschlichen X3P Microarray.

Beziehen High Quality-RNA aus Einzel-Zellpopulationen in menschlichen Postmortem Gehirngewebe

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

30.5K Aufrufe.  McLean Hospital. Wir beschreiben ein Verfahren mittels Laser-Mikrodissektion zu isolieren und zu extrahieren RNA aus einer homogenen Zellpopulation, pyramidalen Neuronen in der Schicht III des oberen temporalen Gyrus in postmortalen menschlichen Gehirnen. Wir anschließend linear verstärken (T7-based) mRNA und hybridisieren die Probe auf die Affymetrix menschlichen X3P Microarray.

Serie: 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.

Beziehen Eier von Xenopus laevis Weibchen

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, ...

Forschung

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.

Neuronale Kerne Isolation von Human Postmortem Gehirngewebe

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.

Generierung von Einzel-Zellsuspensionen aus Maus Nervengewebe

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.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

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.

Robuste Generierung von Leber-ähnlichen Zellen aus humanen embryonalen Stammzell-Populationen

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.

Generierung von High Quality Chromatin Immunpräzipitation DNA Vorlage für Hochdurchsatz-Sequenzierung (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.

Ein Laser Capture Microdissection Protocol, das hochwertige RNA aus frisch gefrorenen Mausknochen liefert

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.

Quantitativer Proteomik-Workflow mit multipler Reaktionsüberwachung basierender Detektion von Proteinen aus menschlichem Hirngewebe

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.

Gewebeentnahme und RNA-Extraktion aus dem humanen osteoarthritischen Kniegelenk

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.