This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

Todos los animales fueron tratados de acuerdo con los Institutos Nacionales de Salud. El Comité de Cuidado y Uso de Animales de la Universidad de Alabama en Birmingham aprobó el uso de animales. 1. La obtención de ARN y ADN de un Enriquecido astrocíticos Población que utiliza fluorescente clasificación de células activadas (FACS) de astrocitos del tejido cerebral entera <ol><li> Animales tranquilo con CO 2 durante 1 minuto y luego decapitar rápidamente. Diseccionar cortezas como se describe en Albuquerque et al, 26.; no es necesario para eliminar meninges. Nota: Las ratas transgénicas que expresan eGFP bajo la S100β (un marcador de astrocitos) promotor se generaron por Itakura et al 27 y se utilizan para FACS clasificación de los astrocitos.. </li><li> Preparar homogeneizado de todo el cerebro usando un kit de disociación de papaína bajo condiciones no estériles de acuerdo con el protocolo del fabricante. Calor a activar la papaína a 37 ° C en baño de agua durante 10 minutos. Nota:Solución de papaína es proporcionada por el fabricante y contiene L-cisteína y EDTA (Ver Tabla de Materiales). <ol><li> Cortar o dremel un agujero en la parte superior de un tubo cónico de 50 ml para permitir que la tubería que lleva 95% O 2: 5% de CO 2 que se introduce en un tubo cónico de cerrada (figura 1A). Coloque cortezas disecados en 10 mm placa de cultivo que contienen medios de disociación (MEM suplementado con glucosa 20 mM y penicilina / estreptomicina (500 U / ml) y se equilibró a 95% O 2: 5% de CO 2) y el uso de una hoja de afeitar limpia para picar el tejido en 1 x 1 mm 2 piezas. </li></ol></li><li> Tejido de transferencia utilizando 10 ml Pipetman manual de 50 ml tubo cónico que contienen solución de papaína. Permitir que el tejido se asiente a la parte inferior de la pipeta de transferencia antes de la descarga para minimizar la cantidad de medios de disociación prorrogados. Mantenga solución de papaína se equilibró a 95% O 2: 5% de CO 2 a través de la superficie de intercambio de gases durante la duración de la incubación. No burbuja papaína solutien. Incubar el tejido durante 20 minutos en 37 ° C baño de agua. </li><li> Después de la incubación en solución de papaína, el tejido triturado 10 veces con una pipeta 10 ml de transferencia a baja velocidad. Centrifugar la suspensión celular nublado en 1000 xg durante 5 min a TA. <ol><li> Equilibrar solución de inhibidor de ADNasa / albúmina (proporcionado por el fabricante) a través de intercambio de gas de la superficie y volver a suspender las células sedimentadas en 3 ml de solución de inhibidor / albúmina de DNasa. Preparar gradiente comercial de densidad discontinuo, siguiendo las instrucciones del fabricante. </li></ol></li><li> Girar gradiente de densidad discontinua en 1000 xg durante 6 min. Aislar las células disociadas a partir de la parte inferior del tubo por la succión pellet parte inferior usando una pipeta. </li><li> Vuelva a suspender las células disociadas en 2-3 ml de DPBS con 0,02% de albúmina de suero bovino y 1 mg / ml de DNasa o HEPES preferidos tamponadas medios de cultivo. Pasar a través de 40 micras filtro antes de FACS (Figura 2A). Mantener las células en hielo hasta la clasificación. <ol><li> Realizar FACS 28. &lt;/ li&gt; <li> Células de pellets de 1,5 ml tubos de centrifugación a 2.000 xg durante 5 min a 4 ° C. Nota: los astrocitos sedimentaron se pueden utilizar inmediatamente o mantenerse en -80 ° C hasta la extracción de ADN. </li></ol></li><li> Extracto de ARN y el ADN utilizando el método de aislamiento preferido 29 30. Evaluar ARN y la concentración de ADN y la calidad a través de espectrofotómetro y bioanalizador 31. Nota: Utilizar únicamente ARN de alta calidad y de ADN en los pasos siguientes, 260/280 = 2,0 a 2,2 y 1,8 a 1,9, respectivamente. Análisis Bioanalyzer es esencial para evaluar la degradación del ARN o la fragmentación del ADN. ARN o ADN pueden ser utilizados inmediatamente o almacenados en -80 ° C o -20 ° C, respectivamente, para estudios posteriores. </li></ol> 2. La evaluación de metilación del ADN de estado de un gen utilizando Análisis Melt metilación sensible a alta resolución (MS-HRMA) <ol><li> Introduzca la secuencia del gen de interés en software de mapas de metilación en línea preferido para identificar cualquier islas CpG en el gende interés 32. <ol><li> Diseño cebadores contra bisulfito convierten secuencia de ADN 33 y amplifican utilizando ADN polimerasa preferida de acuerdo con el protocolo del fabricante. Verificar el tamaño del producto amplificado mediante la ejecución de un gel de agarosa al 1% de ADN a 100 V durante 45 min. Cebadores Tienda en concentración madre de 20 micras de -20 ° C. </li></ol></li><li> Bisulfito convertir 500-1.000 ng de ADN de cada muestra y de ADN metilado normas que van desde 0 hasta 100% de la misma especie animal 34. Eluir las muestras para proporcionar una concentración de 20 ng / l. Verifique concentración de ADN convertida bisulfito mediante espectrofotómetro 31. </li><li> Configuración 20 reacciones mu l para la amplificación de MS-GRH usando ADN polimerasa preferido y cebadores a las 5 de la concentración micras, de conformidad con la Tabla 1 y 2. Ejecute todas las muestras, incluyendo ADN y metilados normas FACS, por triplicado. </li><li> Dependiendo de software de análisis, ajuste antes y después de iniciar y detener parámetrosalrededor de las transiciones de la curva de fusión. Set de inicio pre-fusión y los parámetros de parada antes de la fusión en lo que la diferencia entre los dos es desde 0,2 hasta 0,5 ° C. Establecer inicio posterior a la fusión y posterior a la fusión detener de manera similar. Extraer datos de diferencia de temperatura máxima para cada muestra. </li><li> El uso de estándares ciento metilados (y-valor) y sus correspondientes diferencias de temperatura pico promedio (x-valor) generar una ecuación de regresión lineal (Figura 3A-B, Tabla 3.4). Utilice esta ecuación de regresión lineal para estimar el estado de metilación de muestras desconocidas 35. </li></ol> 3. Evaluar-metilado Hyper Promotor Actividad través Uso de ensayo de luciferasa <ol><li> Identificar las islas CpG de gen diana (Paso 2.1). Amplificar por PCR las regiones de interés 36 y el clon aguas arriba del gen informador de la luciferasa de la luciérnaga luc2 para producir plásmidos CpG-island-luc2 37. </li><li> Linealizar 30 g de plásmido CpG isla luc2 través de la restriccióndigestión con enzimas de 38. Verificar los sitios de digestión de restricción y evitar cortes dobles utilizando el software de corte preferido 38. Heat-inactivar enzimas a temperatura y duración adecuada digestión siguientes de acuerdo con el protocolo del fabricante. Pérdida mínima de ADN se produce durante el paso de linealización. </li><li> Metilato plásmidos linealizadas utilizando CpG metilasa (M.Sssl) O / N a 30 ° C o dejar sin tratar fabricante de protocolo siguiente a excepción de los siguientes ajustes. </li><li> Realizar 50 reacciones mL. </li><li> Utilice 5 unidades de CpG metilasa para metilar 700 ng de plásmido linealizado. </li><li> Ejecute reacciones O / N de 13 a 19 h. </li><li> Después de la reacción metilasa de CpG, realice la limpieza de ADN utilizando estándar, comercialmente membrana de gel de sílice disponibles limpiar kit de acuerdo con el protocolo del fabricante. Pérdidas significativas de ADN ocurren siguiente reacción metilasa de CpG, la pérdida de 30 a 60%. </li><li> Verificar la metilación de los plásmidos a través de una digestión de restricción Hpa II. </li&gt; <li> Tome 1 g de ADN metilado o no metilado y digestión de restricción con Hpa II durante 1 hora a 37 ° C. Utilice 5-10 unidades de Hpa II para cada g de ADN. Después de la digestión Hpa II, realice la limpieza de ADN utilizando preferido membrana disponible comercialmente de gel de sílice limpiar kit. Ejecutar ambos plásmidos metiladas y no metiladas CpG DNA en un gel de agarosa al 1% en tampón TAE o TBE a 100 V durante 45 min para la visualización (Figura 4B). </li><li> Asunto tanto metilado y no metilado plásmidos a doble digestión con enzimas de restricción apropiadas para liberar de larga duración luc 2 (vector) y la isla CpG (inserto) fragmentos 38. Realice doble digestión O / N a la temperatura adecuada y enzimas que inactivan calor después de la digestión de acuerdo con el protocolo del fabricante. Mínima pérdida de la concentración de ADN se produce durante la restricción doble digestión. </li><li> Ejecutar plásmidos digeridos doble sobre 1% gel de agarosa de ADN a 100 V durante 1 h para permitir la separación of vector e inserte (Figura 4C). Precaución. El uso de protección UV protector de cara, coloque gel de ADN en una luz negro mesa para visualizar las bandas. Basado en el tamaño, el consumo de inserción metilado y no metilado y no metilado vector utilizando una cuchilla quirúrgica limpia. </li><li> Gel de ADN usando extracto de fenol saturado, pH 6,6. En pocas palabras, pesar gel de ADN que contiene vector o inserto. Utilice 100 l de fenol por 0,1 gramos de gel de ADN. Homogeneizar gel de ADN en fenol utilizando homogeneizador Dounce vidrio. </li><li> Añadir cloroformo (usando un quinto del volumen de fenol) y agitar muestras durante 20 s. Incubar las muestras a temperatura ambiente durante 2-3 min. Centrifugar durante 15 minutos a velocidad máxima (16,1 x 1000 x g) a 4 ° C. </li><li> Retire la solución acuosa y añadir volumen 0,1X de 3 M de acetato de sodio y el volumen 2,5X de etanol. Incubar las muestras durante 1 hora a -80 ° C. Después de la incubación, retirar el etanol y resuspender el ADN en 30 l de tampón preferido. Pérdida significativa de ADN se produce después del aislamiento de insertos y vectores, el 30-50% loss. </li><li> Re-ligar las inserciones metilados y no metilados a vector no metilado utilizando ADN ligasa de T4 38. Utilice una proporción de 1: 4 de vector para insertar para reacciones de ligación. Reacción de instalación de acuerdo con las instrucciones del fabricante. Use un volumen total de 50 l para las reacciones e incubar O / N a -20 ° C o en hielo con tapa sobre el cubo de hielo. </li><li> Después de la ligación, realice la limpieza de ADN utilizando preferido membrana disponible comercialmente de gel de sílice limpiar kit. Pérdida significativa de ADN ocurrir después de reacción de ligación, un aproximado de la pérdida de 30 a 50% de ADN. Verificar re-ligación mediante la ejecución en 1% de gel de agarosa de ADN a 100 V durante 45 min y evaluar la concentración de plásmidos re-ligado (Figura 4D). </li><li> Transfectar plásmidos metiladas o no metiladas en células D54 usando un reactivo de transfección comercial según el protocolo del fabricante. Células D54 semilla sobre la placa de 12 pocillos en un 0.14 x 10 6 células / pocillo. </li><li> Después de 24 horas, las células transfectar wiconcentraciones iguales th de cualquiera de (1) plásmido no methyated CpG-luc2 + Renilla luciferasa u otro vector de control o (2) metilado CpG-plásmido luc2 + Renilla luciferasa u otro vector de control. </li><li> Permitir a las células para transfectar durante 24 horas antes de realizar el ensayo de luciferasa dual. Realizar ensayo de acuerdo con las instrucciones del fabricante. Realizar lecturas utilizando un luminómetro. Lea cada pocillo por triplicado. </li><li> Calcular la relación de la actividad luciferasa de la luciérnaga de Renilla u otra actividad luciferasa control. Normalizar metilado Firefly: actividad luciferasa control para Firefly no metilado: actividad de la luciferasa de control al dividir la actividad luciferasa metilado por la actividad de la luciferasa no metilado </li></ol>

The authors have no disclosures.

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i...

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Primer</td>
			<td>Applied Biosystems</td>
			<td>online</td>
			<td>software to localize CpG Islands</td>
		</tr>
		<tr>
			<td>EZ DNA methylation Kit</td>
			<td>Zymo Research</td>
			<td>D5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Premixed Calibration Standard</td>
			<td>EpiGenDx</td>
			<td>80-8060R-Premix</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG Methylase (M.Sssl)</td>
			<td>Zymo Research</td>
			<td>E2010</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction&#160;</td>
			<td>QIagen</td>
			<td>28704</td>
			<td>Used for gel extraction and DNA cleanup</td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td>New England BioLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEB cutter</td>
			<td>New England BioLabs</td>
			<td>online</td>
			<td>verify restriction digest sites</td>
		</tr>
		<tr>
			<td>Dual Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Luc2 vector, pGL4.10</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>renilla vector, pGL4.74</td>
			<td>Promega</td>
			<td>E2241</td>
			<td></td>
		</tr>
		<tr>
			<td>TD-20/20 Luminometer</td>
			<td>Turner Designs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine LTX and Plus Reagent</td>
			<td>Life Technologies</td>
			<td>A12621</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol, saturated pH 6.6/6.9</td>
			<td>Fisher Scientific</td>
			<td>BP 17501-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000/2000c Spectrophtometer</td>
			<td>ThermoScientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MeltDoctor Master Mix</td>
			<td>Life Technologies</td>
			<td>4415440</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Melt (HRM) Software v2.0</td>
			<td>Life Technologies</td>
			<td>4397808</td>
			<td></td>
		</tr>
		<tr>
			<td>AB SDS software v2.3</td>
			<td>Life Technologies</td>
			<td>online</td>
			<td></td>
		</tr>
		<tr>
			<td>AB High Resolution Melting Getting Started Guide&#160;</td>
			<td>Life Technologies</td>
			<td>online</td>
			<td></td>
		</tr>
		<tr>
			<td>AB 7900HT Fast Real-Time System</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Una población enriquecida de astrocitos fue adquirida a través de FACS clasificación de eGFP-S100β animales transgénicos 27. Debido a la disminución de la calidad de las células y moléculas moleculares aisladas de animales mayores de postnatal día 50 (p50), animales p0-p40 edades son óptimas para este tipo de experimentos. Tejido cortical se utilizó para estos experimentos. Cortezas de dos a seis animales se agruparon juntos. FACS se realizó en las instalaciones de la UAB Integral de Citometría d...

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

La correlación de Gene-específica de ADN de metilación cambios con la expresión y actividad transcripcional de astrocíticos<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

correlating-gene-specific-dna-methylation-changes-with-expression

Research

JoVE Journal

Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

7.9K vistas.  University of Alabama at Birmingham. DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. 

Video: Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Investigación

Biología

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determinación de la metilación del ADN de los genes impresos en Arabidopsis Endospermo

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

La metilación del ADN: Bisulfito de modificación y análisis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Perfiles de transcripción de los organismos unicelulares de los cardiomiocitos de ratón adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Un ensayo cuantitativo para estudiar proteínas: DNA Interacciones, reguladores Descubra transcripcional de la expresión génica, e identificar agentes antitumoral

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced reducido Representación bisulfito secuenciación para la Evaluación de la metilación del ADN en la Resolución Base Par

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Dirigida análisis de metilación del ADN mediante secuenciación de próxima generación

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...