Yazarlar Sitometrisi Core tesiste Massachusetts Üniversitesi Tıp Fakültesi Dr. Richard Konz ve personel tarafından sağlanan danışmanlık ve teknik destek için teşekkür ederiz. Diyabet Endokrinoloji Araştırma Merkezi Hibe DK032520 tarafından desteklenen temel kaynakları da kullanılmıştır. Bu çalışma, Ulusal Ruh Sağlığı Enstitüsü (NIMH), Ulusal Uyuşturucu Enstitüsü&#39;nden (NIDA) ve Ulusal Çocuk Sağlığı ve İnsan Gelişimi Enstitüsü hibe tarafından desteklenmektedir.

Çekirdekler Ekstraksiyon <ol><li> Bir douncer 5 ml Lizis Tampon 1, 250 mg donmuş postmortem beyin dokusu koyun ve buz üzerine yerleştirin. Sıralama FACS sonra yaklaşık 5 milyon çekirdekleri almak için, genellikle örnek başına 1000 mg doku kullanın. </li><li> Doku çözülmüş sonra, buz üzerinde ise 1 dakika için doku Dounce. </li><li> Homojenize doku ve 15 ml açık ultrasantrifüjdeki tüp içine yerleştirin. Buz tüpü koyun. </li><li> 9 Sakkaroz Çözüm 2 mL pipet ile alın açık ultrasantrifüjdeki tüpün dibinde pipet koymak ve Sakkaroz Çözüm 2 üstüne homojenize doku çözümü ile bir konsantrasyon degrade oluşturmak için Sakkaroz Çözüm 2 serbest. </li><li> Ultrasantrifüjdeki içinde dönerken tüm dengeli olacağına emin olmak için ultrasantrifüjdeki tüpler tartılır. Lizis Tampon 1, üst tabaka eklenerek ağırlığı herhangi bir ayarlamaları yapın . </li><li> Tüpler tartılır sonra, rotor kova içine koyun. </li><li> SW28 rotor içine yerleştirin ve tüm kovalar rotor ultrasantrifüjdeki dikkatlice yerleştirin. </li><li> 4 2,5 saat süreyle 107163,6 RCF örnekleri ultrasantrifüjdeki ° C </li><li> Tüpün alt kısmındaki çekirdekleri içeren pelet rahatsız etmemek için dikkatli olmak, santrifüj işlemi tamamlandıktan sonra, bir vakum kullanımı ile, konsantrasyon farkı Bulunan enkaz tabakası ile birlikte, süpernatant kaldırmak. </li><li> 1X PBS 500 mcL her pelet ekleyin ve 20 dakika boyunca buz üzerinde bekletin. Bu pelet, daha sonra mekanik stres miktarını azaltır çekirdekleri deneyimi, yukarı ve aşağı pipetleme çözmek için kendi başına çözmek için biraz izin verir. </li><li> Çekirdekleri, 20 dakika boyunca buz üzerinde inkübe sonra, mekanik 1XPBS içinde çekirdekleri tamamen erimesi için aşağı yukarı pipet ve. </li><li> Çekirdekleri içeren çözüm ultrasantrifüjdeki tüpleri çıkarın ve 2 mL tek mikrosantrifüj tüpe iki tüp içeriğini birleştirmek. Yine, buz örnekleri yer. </li></ol> FACS için immün <ol><li> Bu noktada, birincil ve ikincil antikor oluşan bir immün karışımı yapmak ve Engelleme karıştırın. Her bir numune için 300, 1.2 Neun antikor,% 0.5 BSA ve% 10 Normal Keçi 1XPBS Serum ve Alexa Fluor 488 1 mcL içeren Engelleme Mix, 100 mcL mcL içeren 1XPBS mcL birleştirir. Kontrol örnekleri için, birincil antikor Neun dışlamak yerine, sadece daha fazla 1XPBS eklemek. </li><li> Vorteks immün karışımı 5 dakika karanlıkta, oda sıcaklığında ve kuluçkaya yatmaktadır. </li><li> Boyama karışımı inkübe iken, numuneler için kontrollerini yapmak. Yalnız çekirdekleri içeren bir örnek sıralama FACS için, &quot;lekesiz kontrol&quot; tabi olarak adlandırılan ikinci bir örnek, bir negatif kontrol, birincil antikor Neun de tabi olmadan ikincil antikor Alexa Fluor 488 çekirdekleri içeren. </li><li> 980 uL negatif kontrol 1XPBS içeren başka bir 2 ml tüp içine 1XPBS lekesiz kontrolü için, ve başka bir 20 mcL içeren 2 ml mikrosantrifüj tüpe çözüm içeren çekirdeğinin kısım 20 mcL. Hacmi çekirdekleri içeren 1XPBS 1 ml yedeklemek örnek kaldırın. Tüm numuneler, buz üzerinde geri yerleştirilir. </li><li> Immün karışımı inkübe tamamladı Şimdi uygun numuneler 401 mcL içeriğini ekleyin. Olumsuz örnek içeren karışımın sadece ikincil antikor Alexa Fluor 488 alacaksınız çekirdekleri örnek, hem Neun ve Alexa Fluor 488 içeren karışım alacaksınız. </li><li> 45 dakika boyunca karanlıkta döndürmek için soğuk oda içine örnekleri alın. </li><li> Örnekleri 45 dakika boyunca karanlıkta inkübe sonra, buz koyun ve FACS tesisi getirin. FACS tesisine taşıma sırasında, örnekleri, buz üzerinde ve karanlıkta tutulmalıdır. </li></ol> FACS <ol><li> FACS tesiste, 40 um filtresi aracılığıyla numuneler filtre. Sonra, soğuk tutuluyor sıralamadan önce bazı ön verileri elde etmek için, onları bir kaç dakika aracı ile çalışacak. </li><li> Bu noktada, bu sıralama için gerekli olan uygun kapılarını ayarlamak için gereklidir. Birkaç ayrı ayrı kapılardan tür örnekleri için kullanılır. Ilk kapısı örnek içinde bulunan farklı çekirdeğinin boyutu hakkında bir fikir verir ve gerçek çekirdekleri enkaz separatation sağlar. İkinci kapı bizi tek tek çekirdekleri sıralamak için izin verir. Bu noktada atın herhangi bir agrega atılır. Son olarak, üçüncü kapı bizim sekonder antikoru bulundu florokrom bu eşleşen, belirli bir floresan dalga boyu ayarlanır. Bu kapıdan yerleştirilmesi bize düzenlemenize olanak sağlar ve bu nedenle ayrı Neun + Neun nöronal çekirdeklerin çekirdekleri. </li><li> Bütün kapıları seçilmiştir sonra, çekirdekleri 1XPBS sıralanabilir. </li><li> Sonratüm örnek geçti, aracı ile sıralanmış örnek bir kısım çalışan tür saflık kontrol edin. Örnek floresan yayılmamış değil, tek bir kadran içinde yer emin olun. % 90&#39;ın üzerinde saf örnekleri seçmek için en iyisidir. </li></ol> FACS sonra <ol><li> Örnek çekirdekleri işleme başlayabilirsiniz sıralanır olmuştur. 10 ml sıralaması örnek alın ve Sakkaroz Çözüm 2 uL 1M CaCl 2, 50 ve 30 ul 1m Mg (As) 2 2 mL ekleyin. Bir hatırlatma olarak, her zaman örnek çekirdekleri Sabitlenmemiş beri buz üzerinde tutmak için emin olun. </li><li> Az 10 ml sıralaması örnek varsa, 1XPBS ekleyerek 10 ml sesini yükseltmek, ya da ek maddeler buna göre ayarlamak. </li><li> 15 dakika boyunca örnek birkaç kez buz üzerinde ve yerde tersine çevirin. </li><li> , 4, örnekleri 15 dakika boyunca buz üzerinde inkübe sonra 15 dakika boyunca 1786 RCF bir salıncak kova rotor santrifüj ° C </li><li> Borunun alt kısmında bulunan beyaz pelet bozmadan, bir vakum kullanımı ile süpernatant atın. </li><li> 200 ul geçmeden deneyler için gerekli her türlü çözüm pelet çözülür ve pipet ve aşağı yukarı. </li><li> Çözüm küçük bir tüp içine aktarın ve daha sonra kullanmak kadar -80 ° C derin dondurucu içine yerleştirin. </li></ol>

<ol>
	<li>Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., Jaenisch, R., Laird, P. W., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+human+cerebral+cortex+is+dynamically+regulated+throughout+the+life+span+and+involves+differentiated+neurons.">DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.</a> PLoS ONE. 2, 895-895 (2007).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Acevedo, L. G., Iniguez, A. L., Holster, H. L., Zhang, X., Green, R., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+ChIP-chip+analysis+using+10,000+human+cells.">Genome-scale ChIP-chip analysis using 10,000 human cells.</a> Biotechniques. 43, 791-797 (2007).</li></ol>

Burada sunulan protokol daha genel nöronal kromatin epigenetik değişiklikler ve odaklanmış çalışmaları için özellikle yararlı olacaktır, normal gelişmekte olan ve yaşlanma veya nörolojik veya psikiyatrik bir hastalık sırasında nöronal çekirdeği, moleküler fenotipi. Bu yöntem, potansiyel yaşlanma seyri sırasında nöron glia rasyon vardiya veya hastalık nedeniyle beyin dokusunda hücresel heterojenite bir endişe 1 belli bir avantajdır . Örneğin, bugünkü sıralama protokolü kromatin immunoprecipita...

Reaktifler: <table><tr><th colspan="3" align="left"> 1. Lizis Tampon </th></tr><tr><td> 0.32M </td><td> Sakaroz </td><td> 5,47 g </td></tr><tr><td> 5 mM </td><td> CaCl 2 </td><td> 250 ul </td></tr><tr><td> 3 mM </td><td> Mg (Asetat) 2 </td><td> 150 ul </td></tr><tr><td> 0.1 mM </td><td> EDTA </td><td> 10 ul </td></tr><tr><td> 10mM </td><td> Tris-HCl, pH8 </td><td> 500 ul </td></tr><tr><td> 1 mM </td><td> DTT </td><td> 17 ul </td></tr><tr><td> % 0.1 </td><td> Triton X-100 </td><td> 50 ul </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Otoklavlı su ile 50 ml hacim ayarlayın </td></tr></table><table><tr><th colspan="3" align="left"> 2. Sakkaroz Çözüm </th></tr><tr><td> 1,8 M </td><td> Sakaroz </td><td> 30,78 g </td></tr><tr><td> 3 mM </td><td> Mg (Asetat) 2 </td><td> 150 ul </td></tr><tr><td> 1 mM </td><td> DTT </td><td> 17 ul </td></tr><tr><td> 10 mM </td><td> Tris-HCl, pH8 </td><td> 500 ul </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Otoklavlı su ile 50 ml hacim ayarlayın </td></tr></table><table><tr><th colspan="3" align="left"> 3. Tampon Dounce </th></tr><tr><td> 10 mM </td><td> Tris </td><td> 0,242 g </td></tr><tr><td> 4 mM </td><td> MgCl 2 </td><td> 0.163 g </td></tr><tr><td> 1 mM </td><td> CaCl 2 </td><td> 0,03 g </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Otoklavlı su ile 200 ml pH 7.5 ve hacmi ayarlayın. </td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

Insan beyninin nöron prenatal gelişimi sırasında büyük ölçüde postmitotic hale gelir, ve böylece tam ömrü boyunca çekirdekleri korumak. Ancak gelişme ve yaşlanma ders sırasında nöronal kromatin ve nükleer organizasyon değişiklikleri veya kronik nöropsikiyatrik hastalık hakkında bilinen çok az. Ancak bugüne kadar, en Kromatin ve DNA tabanlı testlerin (FISH dışında) eksikliği, tek hücreli çözünürlük. Nöronların genellikle çeşitli alt popülasyonlar glia ve diğer non-nöronal hücrelerin farklı türleri ile iç içe, çünkü bu amaçla, beyin dokusu önemli hücresel heterojenite, önemli bir sınırlama teşkil etmektedir. Hücre türüne özgü kültürleri büyümeye olası bir çözüm olurdu, ama nöronlar da dahil olmak üzere en çok merkezi sinir sistemi hücreleri, sadece birkaç hafta için, en azından, sürdürülebilir ex vivo ve böylece potansiyel olarak tam ömrü boyunca faaliyet epigenetik mekanizmalar için eksik bir model sağlayacak . Burada, (sabit asla) dondurulmuş insan postmortem beyin çekirdeklerinin özü ve arındırmak için bir protokol sağlar. Bu yöntem, anti-neun antikor ile Ultrasantrifügasyon ve immunotagging takip Hipotonik lizis tamponu çekirdeklerinin çıkarılması içerir. Etiketli nöronal çekirdeklerinin daha sonra floresan aktive sıralama kullanılarak ayrı toplanır. Bu yöntem, türlerin geniş bir yelpazede herhangi bir beyin bölgesi için geçerli olup, site ve modifikasyon özgü anti-histon antikorlar ve DNA metilasyon ve diğer testleri ile kromatin immunoprecipitation çalışmalar için uygun olmalıdır.

Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Beyin dokusunda hücresel heterojenite testlerin çoğu tek hücreli çözünürlük eksikliği nedeniyle kromatin epigenetik işaretlerin çalışma için önemli bir sınırlama teşkil etmektedir. Nöronlar genellikle glia ve diğer non-nöronal hücreler ile iç içe. Biz özü ve insan beyninin nöron çekirdekleri toplamak için bir protokol sağlar.

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, ...

neuronal-nuclei-isolation-from-human-postmortem-brain-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.0K Görüntüleme.  University of Massachusetts Medical School. Beyin dokusunda hücresel heterojenite testlerin çoğu tek hücreli çözünürlük eksikliği nedeniyle kromatin epigenetik işaretlerin çalışma için önemli bir sınırlama teşkil etmektedir. Nöronlar genellikle glia ve diğer non-nöronal hücreler ile iç içe. Biz özü ve insan beyninin nöron çekirdekleri toplamak için bir protokol sağlar.

Video: Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

İnsan Beyninin Doku Kromatin Testi

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Biyoloji

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

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Fare ve İnsan Kolon Doku Primer miyofibroblastlar İzolasyonu

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Dan Tagged Çekirdeğin Affinity-tabanlı yalıtım<em&gt; Drosophila</emGen ekspresyon analizi için&gt; Dokular

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Insan serumundan küçük kodlayıcı olmayan RNA&#39;lar izolasyonu

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Fare pankreasından alınan RNA İzolasyonu: A Ribonuclease zengin Doku

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 Yetişkin Cilt Dokularından İnsan Melanositlerin Verimini Zenginleştirir

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Deterjan ve Enzimsiz YöntemLe Tüm Dokudan Çekirdek İzolasyon

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

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.

İnsan Beyin Dokusundan Proteinlerin Çoklu Reaksiyon İzleme Tabanlı Tespiti Kullanılarak Nicel Proteomik İş Akışı

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

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.

İnsan Postmortem Beyin Dokusu Nöronal Çekirdekler İzolasyon

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