Los autores agradecen el asesoramiento y apoyo técnico proporcionado por el Dr. Richard Konz y el personal en las instalaciones de flujo básico de Citometría de la Universidad de Massachusetts Medical School. Los recursos básicos con el apoyo de la Diabetes Endocrinología del Centro de Investigación de Grant DK032520 también fueron utilizados. Este trabajo es apoyado por subvenciones del Instituto Nacional de Salud Mental (NIMH), el Instituto Nacional de Abuso de Drogas (NIDA) y el Instituto Nacional de Salud Infantil y Desarrollo Humano.

Los núcleos de extracción <ol><li> Poner 250 mg de tejido congelado el cerebro post mortem en 5 ml de un tampón de lisis en un douncer y colocarlo sobre hielo. Para recibir unos 5 millones de núcleos después de FACS clasificación, por lo general el uso de 1000 mg de tejido por muestra. </li><li> Una vez que el tejido se ha descongelado, el tejido Dounce durante 1 minuto, mientras que en el hielo. </li><li> Tome el tejido homogeneizado y colocarlo en un tubo de ultracentrífuga 15 ml claro. Ponga el tubo en hielo. </li><li> Tomar 9 mL de solución de sacarosa 2 con una pipeta, poner la pipeta en la parte inferior del tubo de ultracentrífuga claro, y liberar la solución de sacarosa 2 para crear un gradiente de concentración con la solución de tejido homogeneizado en la parte superior de la solución de sacarosa 2. </li><li> Pesar los tubos de ultracentrífuga para asegurarse de que todos ellos serán equilibradas al girar dentro de la ultracentrífuga. Realizar ajustes en el peso mediante la adición de un tampón de lisis a la capa superior. </li><li> Después de que los tubos han sido pesados, colocarlos en cubos del rotor. </li><li> Coloque todos los cubos en un rotor SW28 y coloque cuidadosamente el rotor en la ultracentrifugación. </li><li> Ultracentrífuga las muestras a 107.163,6 RCF durante 2,5 horas a 4 ° C. </li><li> Una vez se haya completado la centrifugación, separar el sobrenadante, junto con la capa de desechos que se encuentran en el gradiente de concentración, con el uso de un vacío, teniendo cuidado de no perturbar el sedimento que contiene los núcleos en la parte inferior del tubo. </li><li> Añadir 500 l de PBS 1X a cada pastilla y deje reposar en hielo durante 20 minutos. Esto permite que la pastilla se disuelva un poco por su cuenta, lo que facilita que se disuelva después mecánicamente pipeteando arriba y hacia abajo, lo que disminuye la cantidad de estrés la experiencia de los núcleos. </li><li> Después de los núcleos que se incubó en hielo durante 20 minutos, mecánicamente pipeta hacia arriba y abajo, para disolver completamente los núcleos dentro de 1XPBS. </li><li> Eliminar la solución de los núcleos que contienen los tubos de ultracentrífuga y combinar el contenido de dos tubos en un solo tubo de microcentrífuga de 2 ml. Una vez más, el lugar de las muestras en hielo. </li></ol> La inmunotinción para FACS <ol><li> En este punto, hacer una mezcla compuesta por inmunotinción de anticuerpos primaria y secundaria, y el bloqueo de la mezcla. Para cada muestra, se combinan 300 L de 1XPBS que contiene 1,2 l de anticuerpos NeuN, 100 l de mezcla de bloqueo, que contiene 0,5% de BSA y el 10% suero normal de cabra en 1XPBS, y 1 l de Alexa Fluor 488. Para las muestras de control, excluye la NeuN anticuerpo primario y, en cambio, sólo tiene que añadir más 1XPBS. </li><li> Vortex la mezcla inmunotinción e incubar durante 5 minutos a temperatura ambiente en la oscuridad. </li><li> Mientras que la mezcla de tinción se está incubando, que los controles de las muestras. Por FACS clasificación, una muestra que contiene núcleos solo, llamado el &quot;control sin mancha&quot; es necesario, una segunda muestra, un control negativo, que contiene el núcleo con el anticuerpo secundario Alexa Fluor 488 sin NeuN anticuerpo primario es también necesaria. </li><li> Alícuota de 20 l de los núcleos que contiene la solución en un tubo de microcentrífuga de 2 ml contiene 1XPBS para el control de mancha, y otro 20 l en otro tubo de 2 ml que contiene 980 l de 1XPBS para el control negativo. Aumentar el volumen de los núcleos que contienen una copia de seguridad muestra a 1 ml con 1XPBS. Todas las muestras se colocan de nuevo en hielo. </li><li> Ahora que la mezcla de inmunotinción ha terminado la incubación, añadir su contenido de 401 l en la toma de muestras adecuadas. La muestra de núcleos recibirá la mezcla que contiene tanto NeuN y Alexa Fluor 488, mientras que la muestra negativa recibirá la mezcla que sólo contenga el anticuerpo secundario Alexa Fluor 488. </li><li> Tomar las muestras en la cámara frigorífica para girar en la oscuridad durante 45 minutos. </li><li> Después de las muestras se han incubado en la oscuridad durante 45 minutos, los ponen en hielo y llevarlos al centro de FACS. Durante el transporte a las instalaciones de la FACS, las muestras deben mantenerse en hielo y en la oscuridad. </li></ol> FACS <ol><li> En las instalaciones de la FACS, filtro de las muestras a través de un filtro de 40 um. A continuación, ejecutar a través del instrumento durante unos minutos, mientras que se mantiene frío, con el fin de obtener algunos datos preliminares antes de la clasificación. </li><li> En este punto, es necesario establecer las puertas apropiadas requeridas para la clasificación. Varias puertas se utilizan diferentes a las muestras de clase. La primera puerta da una idea del tamaño de los núcleos diferentes que se encuentran dentro de la muestra, y permite separatation de escombros de los núcleos actuales. La segunda puerta que nos permite ordenar los núcleos de forma individual. Deseche todos los agregados se descartan en este momento. Por último, la tercera puerta está establecido en una longitud de onda de fluorescencia específica, igual a la del fluorocromo en nuestro anticuerpo secundario. La colocación de esta puerta que nos permite clasificar y separar NeuN +, por lo tanto, los núcleos neuronales de NeuN - núcleos. </li><li> Después de todas las puertas son elegidos, los núcleos se pueden clasificar en 1XPBS. </li><li> Una vez que eltotalidad de la muestra ha pasado, comprobar la pureza de la especie mediante la ejecución de una parte alícuota de la muestra ordenados a través del instrumento. Asegúrese de que la fluorescencia de la muestra no es hacia fuera, sino que está contenido en un solo cuadrante. Lo mejor es elegir las muestras que son más del 90% de pureza. </li></ol> Después de FACS <ol><li> Una vez que la muestra ha sido ordenada, el procesamiento de los núcleos puede comenzar. Tomar 10 ml de muestra ordenados y añadir a 2 ml de solución de sacarosa 2, 50 uL de 1 M de CaCl 2 y 30 ul de 1m Mg (As) 2. Como recordatorio, siempre asegúrese de mantener la muestra en hielo desde los núcleos son fijadas. </li><li> Si hay menos de 10 ml de muestra ordenada, subir el volumen hasta 10 ml mediante la adición de 1XPBS, o ajustar los materiales adicionales en consecuencia. </li><li> Invertir la muestra en varias ocasiones y el lugar en hielo durante 15 minutos. </li><li> Después de que las muestras han incubaron en hielo durante 15 minutos, los de centrifugación en un rotor basculantes a 1786 rcf durante 15 minutos a 4 ° C. </li><li> Eliminar el sobrenadante con el uso de un vacío, sin perturbar el precipitado blanco en la parte inferior del tubo. </li><li> Disolver el precipitado en 200 l de cualquier solución que usted requiere para los experimentos de procedimiento, y la pipeta hacia arriba y hacia abajo. </li><li> La transferencia de la solución en un tubo más pequeño y colocarlo en el congelador de -80 º C hasta su uso posterior. </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>

El protocolo que aquí se presenta debe ser particularmente útil para los estudios se centraron en los cambios epigenéticos de la cromatina neuronal y, en general, sobre el fenotipo molecular del núcleo neuronal, durante el funcionamiento normal desarrollo de la enfermedad y el envejecimiento, o neurológicos o psiquiátricos. El método es particularmente ventajoso cuando la heterogeneidad celular del tejido cerebral, incluyendo posibles cambios en la ración de las neuronas a células gliales en el transcurso del tiempo ya causa de la enfermedad son una preocupación 1. Por ejemplo, al combinar el protocolo de clasificación se presentan con las técnicas de inmunoprecipitación de la cromatina, lo describió recientemente neuronal específica patrones de metilación de las histonas en el factor neurotrófico derivado del cerebro (BDNF), promotor del gen, y 2 para otros genes neuronales. Un método similar se ha usado previamente para recoger las neuronas de la corteza cerebral de adultos para el nacimiento de retrospectivas que data 3. La técnica de clasificación se empleó también en cerebro de ratón 2, y debido a la estabilidad de los núcleos de congelado y descongelado el tejido luego, esperamos que el enfoque que aquí se presenta es aplicable a una amplia gama de especies. Debido a la inmunoprecipitación de la cromatina de tan sólo 10.000 células por ahora es factible incluso para una cobertura más completa del genoma 4, puede ser posible utilizar mucho menor que la de 1000 mg de material de partida que se detalla más arriba.

Reactivos: <table><tr><th colspan="3" align="left"> 1. Tampón de lisis </th></tr><tr><td> 0,32 M </td><td> Sacarosa </td><td> 5,47 g </td></tr><tr><td> 5 mM </td><td> CaCl2 </td><td> 250 l </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 l </td></tr><tr><td> 0,1 mM </td><td> EDTA </td><td> 10 l </td></tr><tr><td> 10 mM </td><td> Tris-HCl, pH 8 </td><td> 500 l </td></tr><tr><td> 1 mM </td><td> TDT </td><td> 17 l </td></tr><tr><td> 0,1% </td><td> Triton X-100 </td><td> 50 l </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajuste el volumen a 50 ml con agua esterilizada </td></tr></table><table><tr><th colspan="3" align="left"> 2. Solución de sacarosa </th></tr><tr><td> 1,8 M </td><td> Sacarosa </td><td> 30,78 g </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 l </td></tr><tr><td> 1 mM </td><td> TDT </td><td> 17 l </td></tr><tr><td> 10 mM </td><td> Tris-HCl, pH 8 </td><td> 500 l </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajuste el volumen a 50 ml con agua esterilizada </td></tr></table><table><tr><th colspan="3" align="left"> 3. Dounce buffer </th></tr><tr><td> 10 mM </td><td> Tris </td><td> 0,242 g </td></tr><tr><td> 4 mM </td><td> MgCl2 </td><td> 0,163 g </td></tr><tr><td> 1 mM </td><td> CaCl2 </td><td> 0,03 g </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajustar el pH a 7.5 y el volumen a 200 ml con agua esterilizada </td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

Las neuronas en el cerebro humano se postmitóticas en gran parte durante el desarrollo prenatal, y así mantener sus núcleos en todo el ciclo de vida completo. Sin embargo, poco se sabe acerca de los cambios en la cromatina nuclear y la organización neuronal durante el curso del desarrollo y el envejecimiento, o en las enfermedades neuropsiquiátricas crónicas. Sin embargo, hasta la fecha la mayoría de los ensayos basados ​​en la cromatina y el ADN (excepto los de pescado) Resolución de la falta de células individuales. Con este fin, la gran heterogeneidad celular del tejido cerebral supone una limitación importante, ya que por lo general diversas subpoblaciones de neuronas se entremezclan con los diferentes tipos de glía y otras células no neuronales. Una posible solución sería hacer crecer células de tipo culturales específicas, pero la mayoría de las células del SNC, incluyendo las neuronas, son ex vivo sostenible, en el mejor de los casos, sólo un par de semanas y por lo tanto proporcionaría un modelo incompleto de los mecanismos epigenéticos potencialmente operan en todo el ciclo de vida completo . Aquí, ofrecemos un protocolo para extraer y purificar los núcleos de congelación (no fijos) del cerebro de cadáver. El método consiste en la extracción de núcleos en solución amortiguadora de lisis hipotónica, seguida por ultracentrifugación y immunotagging con anticuerpos anti-NeuN. Núcleos etiquetados neuronal continuación, se recogen por separado mediante fluorescencia activadas por la clasificación. Este método debe ser aplicable a cualquier región del cerebro en una amplia gama de especies y adecuada para los estudios de inmunoprecipitación de la cromatina en el sitio-y la modificación de los anticuerpos específicos anti-histonas, y por la metilación del ADN y otros ensayos.

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

La heterogeneidad celular del tejido cerebral supone una limitación importante para el estudio de las marcas epigenéticas en la cromatina, porque la mayoría de los ensayos de la falta de resolución única célula. Las neuronas normalmente se entremezclan con la glía y otras células no neuronales. Ofrecemos un protocolo para extraer y recoger los núcleos neuronales del cerebro humano.

Aislamiento de los núcleos neuronales a partir de tejido cerebral humano post-mortem

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 vistas.  University of Massachusetts Medical School. La heterogeneidad celular del tejido cerebral supone una limitación importante para el estudio de las marcas epigenéticas en la cromatina, porque la mayoría de los ensayos de la falta de resolución única célula. Las neuronas normalmente se entremezclan con la glía y otras células no neuronales. Ofrecemos un protocolo para extraer y recoger los núcleos neuronales del cerebro humano.

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 ensayo de la cromatina de tejido cerebral humano

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

Investigación

Biología

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.

La obtención de ARN de alta calidad a partir de poblaciones de células individuales en tejido cerebral humano post-mortem

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.

Aislamiento de Miofibroblastos primarias de ratón y el tejido del colon humano

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.

Aislamiento a base de afinidad de Tagged Núcleos de<em&gt; Drosophila</em&gt; Los tejidos para análisis de expresión génica

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.

El aislamiento de los pequeños RNAs no codificantes de suero humano

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.

Aislamiento de ARN de ratón Páncreas: Un tejido de ribonucleasa-rica

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 enriquece el rendimiento de los melanocitos humanos de los tejidos adultos de la piel

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.

Aislamiento de núcleos de tejido entero utilizando un método de detergente y libre de enzimas

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.

Flujo de trabajo de proteómica cuantitativa utilizando la detección basada en el monitoreo de reacciones múltiples de proteínas del tejido cerebral humano

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.