Gli autori apprezzano la consulenza e il supporto tecnico fornito dal Dr. Richard Konz e del personale presso l&#39;impianto di base Citometria a flusso presso l&#39;Università del Massachusetts Scuola Medica. Risorse di base sostenuto dalla Endocrinologia Diabetes Research Center di Grant DK032520 sono stati utilizzati anche. Questo lavoro è supportato anche da finanziamenti del National Institute of Mental Health (NIMH), l&#39;Istituto Nazionale di Drug Abuse (NIDA) e il National Institute of Child Health e lo Sviluppo Umano.

Nuclei di estrazione <ol><li> Mettere 250 mg di tessuto cerebrale post-mortem congelato in 5 ml di Lysis Buffer 1 in douncer e posizionarlo sul ghiaccio. Per ricevere circa 5 milioni di nuclei dopo FACS ordinamento, useremo 1000 mg di tessuto per campione. </li><li> Una volta che il tessuto si è scongelato, dounce il tessuto per 1 minuto, mentre sul ghiaccio. </li><li> Prendete il tessuto omogeneizzato e posizionarlo in un tubo da 15 ml ultracentrifuga chiaro. Mettere la provetta in ghiaccio. </li><li> Prendere 9 mL di soluzione di saccarosio 2 con una pipetta, mettere la pipetta sul fondo della provetta ultracentrifuga chiaro, e rilasciare la soluzione di saccarosio 2 per creare un gradiente di concentrazione con la soluzione del tessuto omogeneizzato in cima alla soluzione di saccarosio 2. </li><li> Pesare i tubi ultracentrifuga per assicurarsi che saranno tutti bilanciati durante la rotazione all&#39;interno del ultracentrifuga. Apportare eventuali modifiche al peso con l&#39;aggiunta di Lysis Buffer 1 al livello superiore. </li><li> Dopo che i tubi sono stati pesati, li posto in secchi del rotore. </li><li> Mettere tutti i secchi in un rotore SW28 e con attenzione il rotore posto in ultracentrifuga. </li><li> Ultracentrifuga i campioni a 107.163,6 RCF per 2,5 ore a 4 ° C. </li><li> Una volta che la centrifugazione, rimuovere il supernatante, insieme con lo strato di detriti trovati al gradiente di concentrazione, con l&#39;uso di un vuoto, facendo attenzione a non disturbare il pellet contenente i nuclei sul fondo della provetta. </li><li> Aggiungere 500 ml di PBS 1X a ciascun pellet e lasciate riposare in ghiaccio per 20 minuti. Questo permette al pellet per sciogliere un po &#39;da sola, rendendo più facile per poi meccanicamente sciogliere pipettando su e giù, che riduce la quantità di stress dell&#39;esperienza nuclei. </li><li> Dopo i nuclei sono incubati in ghiaccio per 20 minuti, meccanicamente pipetta su e giù per sciogliere completamente i nuclei all&#39;interno 1XPBS. </li><li> Rimuovere i nuclei contenenti soluzione dai tubi ultracentrifuga e combinare il contenuto di due tubi in un unico tubo di 2 ml microcentrifuga. Ancora una volta, posto i campioni sul ghiaccio. </li></ol> Immunocolorazione per FACS <ol><li> A questo punto, fare una miscela composta immunocolorazione di anticorpi primari e secondari, e blocco Mix. Per ogni campione, unire 300 ml di 1XPBS contenente 1,2 ml di anticorpi NeuN, 100 ml di miscela di blocco, che contiene 0,5% di BSA e il 10% siero di capra normale in 1XPBS, e 1 ml di Alexa Fluor 488. Per i campioni di controllo, escludere la NeuN primario di anticorpi e, invece, è sufficiente aggiungere altre 1XPBS. </li><li> Vortex la miscela immunocolorazione e incubare per 5 minuti a temperatura ambiente al buio. </li><li> Mentre la miscela colorazione è in incubazione, fanno i controlli per i campioni. Per FACS ordinamento, un campione, che contiene nuclei sola, chiamata &quot;controllo senza macchia&quot; è necessario, un secondo campione, un controllo negativo, che contiene i nuclei con l&#39;anticorpo secondario Alexa Fluor 488 senza il NeuN anticorpo primario è anche necessario. </li><li> Aliquota 20 l di nuclei che contiene la soluzione in una provetta contenente 2 ml 1XPBS per il controllo senza macchia, e un altro 20 l in un altro tubo da 2 ml contenente 980 uL di 1XPBS per il controllo negativo. Aumentare il volume dei nuclei contenenti campioni di nuovo fino a 1 ml con 1XPBS. Tutti i campioni sono posti di nuovo sul ghiaccio. </li><li> Ora che la miscela immunocolorazione ha finito di incubazione, aggiungere il suo contenuto di 401 microlitri in campioni appropriati. Il campione nuclei riceverà la miscela contenente sia NeuN e Alexa Fluor 488, mentre il campione negativo riceverà la miscela contenente solo l&#39;anticorpo secondario Alexa Fluor 488. </li><li> Prendete i campioni nella stanza fredda per ruotare al buio per 45 minuti. </li><li> Dopo che i campioni sono incubati al buio per 45 minuti, metterli su ghiaccio e portarli alla struttura FACS. Durante il trasporto verso l&#39;impianto FACS, i campioni devono essere tenuti sul ghiaccio e al buio. </li></ol> FACS <ol><li> Presso l&#39;impianto di FACS, filtrare il campione attraverso un filtro di 40 um. Poi, li attraversano lo strumento per pochi minuti, pur essendo tenuti a freddo, al fine di ottenere alcuni dati preliminari prima della cernita. </li><li> A questo punto, è necessario impostare le porte appropriate richieste per l&#39;ordinamento. Diverse porte differenti sono usati per i campioni sorta. Il primo cancello dà un&#39;idea delle dimensioni dei nuclei diversi trovato all&#39;interno del campione, e permette separatation di detriti da nuclei reali. La seconda porta ci permette di ordinare i nuclei singolarmente. Scartare qualsiasi aggregati vengono scartate a questo punto. Infine, la terza porta è impostata su una specifica lunghezza d&#39;onda di fluorescenza, equivalente a quella del fluorocromo trovato nel nostro anticorpo secondario. Ponendo questa porta ci permette di ordinare e + NeuN separati, quindi nuclei neuronali da NeuN - nuclei. </li><li> Dopo che tutti i cancelli sono stati scelti, i nuclei possono essere ordinate in 1XPBS. </li><li> Una volta che ilintero campione è passato attraverso, verificare la purezza del genere eseguendo una aliquota del campione ordinati attraverso lo strumento. Assicurarsi che la fluorescenza del campione non si sviluppa, ma è contenuta all&#39;interno di un quadrante singolo. E &#39;meglio scegliere i campioni che sono oltre il 90% puro. </li></ol> Dopo FACS <ol><li> Una volta che il campione è stato ordinato, il trattamento dei nuclei può cominciare. Prendere 10 ml di campione ordinati e aggiungere ad esso 2 mL di soluzione di saccarosio 2, 50 uL di 1M CaCl 2 e 30 ul di 1m Mg (Ace) 2. Come promemoria, sempre essere sicuri di mantenere il campione in ghiaccio in quanto i nuclei sono non fissata. </li><li> Se c&#39;è meno di 10 ml di campione ordinati, alzare il volume fino a 10 ml con l&#39;aggiunta di 1XPBS, o regolare il materiale aggiuntivo di conseguenza. </li><li> Invertire il campione più volte e posto in ghiaccio per 15 minuti. </li><li> Dopo che i campioni sono incubati in ghiaccio per 15 minuti, le centrifughe in un rotore swing a 1786 RCF per 15 minuti a 4 ° C. </li><li> Eliminare il supernatante con l&#39;uso di un vuoto, senza disturbare il pellet bianco trovato sul fondo della provetta. </li><li> Sciogliere il pellet in 200 ml di soluzione di qualunque avete bisogno per gli esperimenti di procedere, e pipetta su e giù. </li><li> Trasferire la soluzione in un tubo più piccolo e metterlo nel congelatore ° -80 C fino a quando un ulteriore uso. </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>

Il protocollo presentato qui dovrebbe essere particolarmente utile per gli studi focalizzati sui cambiamenti epigenetici della cromatina neuronale e, più in generale, sul fenotipo molecolare del nucleo neuronale, durante il normale sviluppo delle malattie e l&#39;invecchiamento, o neurologici o psichiatrici. Il metodo è particolarmente vantaggioso quando l&#39;eterogeneità cellulare del tessuto cerebrale, tra cui i potenziali cambiamenti nei neuroni-to-glia razione durante il corso di invecchiamento oa causa di malattia sono un problema 1. Per esempio, combinando il protocollo attuale ordinamento con le tecniche di immunoprecipitazione della cromatina, abbiamo recentemente descritto neuroni specifici pattern di metilazione degli istoni il cervello derivato fattore neurotrofico (BDNF), promotore del gene, e per altri geni neuronali 2. Un metodo simile era stato usato precedentemente per raccogliere i neuroni dalla corteccia cerebrale per adulti nascita retrospettiva datazione 3. La tecnica di selezione è stato impiegato anche nel cervello del mouse 2, e per la stabilità dei nuclei in congelati-then-tessuto scongelato, prevediamo che l&#39;approccio qui presentato è applicabile a una vasta gamma di specie. Perché immunoprecipitazione della cromatina da un minimo di 10.000 cellule per ora è fattibile anche per una copertura più completa del genoma 4, può essere possibile utilizzare molto meno di 1000 mg di materiale di partenza che si consiglia di sopra.

Reagenti: <table><tr><th colspan="3" align="left"> 1. Lysis Buffer </th></tr><tr><td> 0.32M </td><td> Saccarosio </td><td> 5,47 g </td></tr><tr><td> 5 mM </td><td> CaCl 2 </td><td> 250 microlitri </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 microlitri </td></tr><tr><td> 0.1 mM </td><td> EDTA </td><td> 10 microlitri </td></tr><tr><td> 10mM </td><td> Tris-HCl, pH8 </td><td> 500 microlitri </td></tr><tr><td> 1 mM </td><td> DTT </td><td> 17 microlitri </td></tr><tr><td> 0,1% </td><td> Triton X-100 </td><td> 50 microlitri </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Regolare il volume a 50 ml con acqua autoclavato </td></tr></table><table><tr><th colspan="3" align="left"> 2. Soluzione di saccarosio </th></tr><tr><td> 1,8 M </td><td> Saccarosio </td><td> 30,78 g </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 microlitri </td></tr><tr><td> 1 mM </td><td> DTT </td><td> 17 microlitri </td></tr><tr><td> 10 mM </td><td> Tris-HCl, pH8 </td><td> 500 microlitri </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Regolare il volume a 50 ml con acqua autoclavato </td></tr></table><table><tr><th colspan="3" align="left"> 3. Dounce tampone </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"> Regolare il pH a 7,5 e il volume a 200 ml con acqua autoclavato </td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

Neuroni nel cervello umano si postmitotic in gran parte durante lo sviluppo prenatale, e quindi mantenere la loro nuclei per tutta la durata della vita piena. Tuttavia, poco si sa sui cambiamenti neuronali e organizzazione della cromatina nucleare durante il corso dello sviluppo e dell&#39;invecchiamento, o in malattie croniche neuropsichiatrici. Tuttavia, ad oggi i test basati la maggior parte della cromatina e DNA (esclusi quelli di pesci) risoluzione mancanza singola cellula. A tal fine, la notevole eterogeneità cellulare del tessuto cerebrale rappresenta una limitazione significativa, perché di solito diverse sottopopolazioni di neuroni sono mescolati con diversi tipi di cellule gliali e di altre cellule non neuronali. Una possibile soluzione sarebbe far crescere specifiche culture di cellule-tipo, ma la maggior parte delle cellule del SNC, tra cui i neuroni, sono ex vivo sostenibile, nella migliore delle ipotesi, solo per poche settimane e quindi avrebbe fornito un modello incompleto per meccanismi epigenetici potenzialmente operanti in tutto il ciclo di vita completo . Ecco, mettiamo a disposizione un protocollo per estrarre e purificare i nuclei da congelati (non fisso) del cervello umano post-mortem. Il metodo prevede l&#39;estrazione dei nuclei in tampone di lisi ipotonica, seguita da ultracentrifugazione e immunotagging con anticorpi anti-NeuN. Etichettati nuclei neuronali vengono poi raccolti separatamente utilizzando la fluorescenza-attivato ordinamento. Questo metodo dovrebbe essere applicabile a qualsiasi regione del cervello in una vasta gamma di specie e adatto per gli studi di immunoprecipitazione della cromatina con il sito-specifici e modifica-anticorpi anti-istoni, e per la metilazione del DNA ed altri test.

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

L&#39;eterogeneità cellulare del tessuto cerebrale rappresenta una limitazione significativa per lo studio delle marcature epigenetiche della cromatina perché la maggior parte saggi mancanza risoluzione singola cella. I neuroni sono in genere mescolati con glia altri e cellule non neuronali. Forniamo un protocollo per estrarre e raccogliere i nuclei neuronali dal cervello umano.

Nuclei di isolamento dal tessuto neuronale del cervello umano Postmortem

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

Nanyang Technological University

Research

JoVE Journal

Biology

22.0K Views.  University of Massachusetts Medical School. L'eterogeneità cellulare del tessuto cerebrale rappresenta una limitazione significativa per lo studio delle marcature epigenetiche della cromatina perché la maggior parte saggi mancanza risoluzione singola cella. I neuroni sono in genere mescolati con glia altri e cellule non neuronali. Forniamo un protocollo per estrarre e raccogliere i nuclei neuronali dal cervello umano.

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.

Un saggio cromatina per tessuto cerebrale umano

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

Ricerca

Biologia

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.

L&#39;ottenimento di RNA di alta qualità da popolazioni di cellule singole nei tessuti umani cervello postmortem

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.

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

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.

L&#39;isolamento a base di affinità di Tagged Nuclei di<em&gt; Drosophila</em&gt; tessuti per 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 ...

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.

Isolamento dei Piccoli RNA non codificanti dal siero umano

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.

Isolamento dell&#39;RNA da mouse Pancreas: Un Tessuto ricco Ribonucleasi

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 arricchisce la resa dei melanociti umani dai tessuti della pelle adulta

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.

Isolamento nuclei dal tessuto intero utilizzando un metodo detergente e senza enzimi

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.

Flusso di lavoro di proteomica quantitativa utilizzando il rilevamento basato sul monitoraggio di reazioni multiple di proteine dal tessuto cerebrale umano

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.