Name: determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts
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HAC fue la Milton E. Cassel de la Rita Allen Foundation (http://www.ritaallenfoundation.org). Este trabajo fue financiado por subvenciones para HAC (P.I. David Botstein), Instituto Nacional de Ciencias de Medicina General del centro de la beca de excelencia P50 GM071508 PhRMA Foundation grant 2007RSGl9572, ciencia nacional Fundación beca OCI-1047879 David agosto, Nacional Institute of General Medical Ciencias R01 GM081686, Instituto Nacional de ciencias médicas General R01 GM0866465, Eli &#38; Edythe Broad Center de medicina regenerativa &#38; investigación con células madre, centro de salud/UCLA CTSI NIH de la mujer Iris Cantor Grant UL1TR000124 y la sociedad de la leucemia linfoma. Investigación en esta publicación fue apoyada por el Instituto Nacional del cáncer de los institutos nacionales de salud bajo la concesión número P50CA092131. Los fundadores no tenían ningún papel en el diseño del estudio, recopilación de datos y análisis, publicación o preparación del manuscrito. HAC es un miembro del Eli &#38; Edythe amplia centro de medicina regenerativa &#38; investigación con células madre, el Instituto de Biología Molecular de la UCLA y el programa interdepartamental de Bioinformática de UCLA.

Todos los experimentos descritos fueron aprobados por comités de revisión institucional en la Universidad de Princeton y la Universidad de California, Los Ángeles.
1. preparar los fibroblastos proliferantes y contacto-inhibido para ActD tiempo curso
Nota: Este protocolo usa una timecourse con cuatro puntos de tiempo. Pueden recogerse tres muestras biológicamente independientes por punto por la recogida de las placas de cultivo de tejidos diferentes en un experimen...

<ol>
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Quietud puede ser inducida por señales externas, incluyendo retiro de mitógenos o suero, falta de adhesión celular y la inhibición de contacto. Inhibición de contacto, uno de varios posibles métodos para inducir la quietud, es un proceso altamente evolutionarily conservado en el que las células de salida ciclo celular proliferativo en respuesta a contacto de célula a célula. Nos centramos aquí en inhibición de contacto como un ejemplo de un método para inducir la quietud. Estudios previos han reportado que co...

Niveles de estado estacionario de transcripciones reflejan la contribución de síntesis transcripción y decaimiento de la transcripción. Regulado y coordinado de la transcripción es un importante mecanismo de control de procesos biológicos1,2,3,4. Por ejemplo, tasas de decaimiento de transcripción han demostrado para contribuir a la serie temporal de acontecimientos después de la activación por el factor de necrosis tumoral citoquinas inflamatorias del5.
Previamente hemos demostrado que la transición entre la proliferación y quietud en fibroblastos humanos primarios se asocia con cambios en los niveles de transcripción de muchos genes6. Algunos de estos cambios reflejan las diferencias en la actividad de factores de transcripción entre estos dos Estados.
Cambios en la tasa de decaimiento de una transcripción también pueden contribuir a cambios en el nivel de expresión de una transcripción en dos diferentes Estados7,8. Basado en los resultados anteriores que la transición entre la proliferación y la inactividad se asocia con cambios en los niveles de varios microRNAs9, le preguntamos si también hay una contribución de la descomposición de la transcripción diferencial de los cambios expresión génica en proliferación contra fibroblastos quiescentes.
Para monitorear la estabilidad de la transcripción, se determinó la tasa de decaimiento de las transcripciones genoma en proliferación versus células quiescentes. Para lograr esto, supervisamos las tasas de decaimiento en proliferación contra fibroblastos quiescentes introduciendo un inhibidor de la transcripción nueva y monitoreo de la tasa en que desaparecieron expedientes individuales con el tiempo. La ventaja de este enfoque es que, en comparación con los métodos que simplemente controlar la expresión génica global, por inhibición de la síntesis nueva de transcripción, seremos capaces de determinar la tasa de decaimiento de estas transcripciones por separado de la tasa a la que son transcrito.
ActD tratamiento para inhibir transcripción nueva y para determinar las tasas de decaimiento de transcripción se ha aplicado con éxito en múltiples estudios anteriores. ActD se ha utilizado para disecar la importancia de la estabilidad del RNA en los cambios en la abundancia de transcripción que resultan del tratamiento con citoquinas proinflamatorias5. Un enfoque similar también se ha utilizado para disecar las diferencias en la tasa de decaimiento de transcripción en proliferación versus células C2C12 diferenciadas como adoptan un fenotipo muscular diferenciado del10. Otro ejemplo, global mRNA vida media también se han detectado en pluripotentes y11de las células madre embrionarias de ratón distinguido. En estos ejemplos, decaimiento del mRNA se ha demostrado para ser importante para la regulación de la abundancia de transcripción y para la transición de células a células diferentes Estados.
Aplicación de los métodos descritos a continuación, descubrimos cambios en las tasas de decaimiento de la transcripción en aproximadamente 500 genes cuando se comparan los fibroblastos en proliferación y quieto Estados12. En particular, hemos descubierto que los objetivos del microRNA miR-29, que es regulada en células quiescentes, se estabilizan cuando las células de transición a la inactividad. Aquí describimos la metodología que se utilizó para determinar las tasas de decaimiento en las células proliferantes y quietas. Esta metodología es útil para comparar las tasas de decaimiento mRNA mundiales en dos distintos pero similares condiciones cuando se busca información sobre genes el decaerse rápidamente. También podría utilizarse para abordar otras cuestiones como el efecto de las condiciones de cultivo celular en decaimiento de la transcripción, por ejemplo, en dos dimensiones versus tres culturas dimensionales. Las tasas de decaimiento pueden ser genoma determinado con métodos tales como microarrays o RNA-seq. alternativamente, qPCR en tiempo real o el borrar norteño puede utilizarse para determinar las tasas de decaimiento en una base de gen por gen o isoforma de la isoforma. Estas tasas pueden utilizarse para calcular la vida media de cada gen supervisada y para identificar genes con índices de caries que son diferentes en dos condiciones.

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			<td height="42" style="height:42px;width:291px;">Centrifuges for microcentrifuge tubes capable of reaching 12,000 x g and 4&#176;C</td>
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			<td height="21" style="height:21px;width:291px;">Equipment for running agarose gels</td>
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			<td height="42" style="height:42px;width:291px;">Sterile tissue culture plates and conical tubes</td>
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			<td height="21" style="height:21px;width:291px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">11965-118</td>
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			<td height="21" style="height:21px;width:291px;">Fetal bovine serum</td>
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			<td height="42" style="height:42px;width:291px;">Sterile serological pipets, pipettors and pipet tips for tissue culture</td>
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			<td height="63" style="height:63px;width:291px;">Individually wrapped, disposable Rnase-free pipettes, pipette tips and tubes for RNA isolation and analysis</td>
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			<td height="42" style="height:42px;width:291px;">Disposable gloves to be worn when handling reagents and RNA samples</td>
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			<td height="42" style="height:42px;width:291px;">RNaseZap</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">AM9780</td>
			<td style="width:237px;">For decontaminating work surfaces from RNase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">2.0 ml eppendorf tubes</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Trypsin-EDTA&#160; Solution 10X</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">9002-07-07</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:291px;">Sterile PBS</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Trizol</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15596018</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Actinomycin D</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">A1410-2 mg</td>
			<td style="width:237px;">inhibits transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sterile DMSO</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">31-761-00ML</td>
			<td style="width:237px;">solvent for actinomycin D</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Chloroform</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN19400225</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Isopropanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP2618500</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP28184</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">RNase-free Glycogen (20 mg/ml aqueous solution)</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0551</td>
			<td style="width:237px;">carrier for RNA precipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:291px;">TURBO DNA-free Kit</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1907</td>
			<td style="width:237px;">removes DNA with DNase, then, in a subsequent step,&#160; inactivates DNA and removes divalent cations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Agarose</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN820721</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Loading dye for RNA gel</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0641</td>
			<td style="width:237px;">suitable even for denaturing electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Millenium RNA markers</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7150</td>
			<td style="width:237px;">RNA ladder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">One-color RNA Spike-in Kit</td>
			<td style="width:163px;">Agilent Technology</td>
			<td style="width:119px;">5188-5282</td>
			<td style="width:237px;">Example spike-in control for Agient microarray analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Tris base</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td style="width:237px;">molecular biology grade, for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Glacial acetic acid</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">A38-500</td>
			<td style="width:237px;">for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">EDTA</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td style="width:237px;">electrophoresis grade, for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethidium bromide</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">E7637</td>
			<td style="width:237px;">for molecular biology</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">External RNA Controls Consortium RNA Spike-in Mix</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td>4456740</td>
			<td style="width:237px;">Spike-in control for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">TruSeq Stranded mRNA Library Preparation Kit A (48 samples, 12 indexes)</td>
			<td style="width:163px;">Illumina</td>
			<td style="width:119px;">RS-122-2101</td>
			<td style="width:237px;">for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">96-well 0.3 ml PCR plate</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AB-0600</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Microseal B adhesive seals</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">MSB1001</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rnase/Dnase-free Reagent Reservoirs</td>
			<td style="width:163px;">VWR</td>
			<td style="width:119px;">89094-662</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Rnase/Dnase-free Eight tube strips and caps</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM12230</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SuperScript Reverse Transcriptase</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">18090010</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">AMPure XP Beads</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
	</tbody>
</table>

determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts

Quietud es un estado temporal, reversible en el que las células han dejado de división celular, pero conservan la capacidad de proliferar. Varios estudios, incluido el nuestro, han demostrado que la quietud se asocia con cambios generalizados en la expresión génica. Algunos de estos cambios se producen a través de cambios en el nivel o la actividad asociada a la proliferación de factores de transcripción, como E2F y MYC. Hemos demostrado que el decaimiento del mRNA también puede contribuir a cambios en la expresión génica entre células proliferantes y quietas. En este protocolo, se describe el procedimiento para el establecimiento de culturas quietas y proliferación de fibroblastos humanos de prepucio cutáneo. A continuación describimos los procedimientos para inhibir la nueva transcripción en las células proliferantes y quietas con actinomicina D (ActD). ActD tratamiento representa un enfoque sencillo y reproducible a disociar la nueva transcripción de la descomposición de la transcripción. Una desventaja del ActD tratamiento es que el curso del tiempo debe ser limitado a un corto plazo porque ActD afecta la viabilidad celular. Niveles de transcripción son monitoreados en el tiempo para determinar las tasas de decaimiento de transcripción. Este procedimiento permite la identificación de genes y de isoformas que exhiben decaimiento diferencial en proliferación contra fibroblastos quiescentes.

Hemos divulgado previamente los resultados de análisis de microarray de las tasas de decaimiento de transcripción en fibroblastos humanos primarios proliferación e inhibida por el contacto durante un tiempo de 8 horas curso12. Se proporciona una lista de genes con un cambio significativo en la estabilidad de la transcripción comparación de fibroblastos proliferantes y contacto-inhibida en la tabla adicional 1. Se proporcionan las intensidades ...

Watch this Scientific Journal Video about Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts at JoVE.com

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Se describe un protocolo para generar la proliferación y fibroblastos dérmicos humanos primarios quietos, seguimiento las tasas de decaimiento de transcripción y la identificación de genes diferencialmente decadentes.

Determinar tasas de decaimiento de transcripción del genoma en quietos y proliferación de fibroblastos humanos

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

The overall goal of this procedure is to monitor transcript decay rates in proliferating and quiescent fibroblasts. This method can help answer key questions in the molecular biology field such as whether changes in gene expression result from changes in transcript decay rate. The main advantage of this technique is that it provides information on transcript decay rate, not just transcript abundance.To begin the protocol, establish proliferating and quiescent cultures of fibroblasts. Proliferating fibroblasts should be trypsinized regularly. Quiescent cultures can be established by allowing plates to reach confluence with regular medium changes.Add ActD 150 microliters to prewarmed medium 10 milliliters for all biological replicate cell culture plates. Add ActD at a concentration of 15 micrograms per milliliter of medium. Mix the solution well.Aspirate the old medium, and add medium containing ActD to the cells. To collect the zero time point sample, gently aspirate off the ActD medium and pipette between five and 10 milliliters of prewarmed PBS onto a 100-millimeter plate, five milliliters of solution for a 100-millimeter plate, two milliliters for a well of a six-well plate, and one milliliter for a well of a 12-well plate. Gently aspirate the PBS.Then add phenol-guanidine isothiocyanate solution directly to the tissue culture plate. Pipette the phenol-guanidine isothiocyanate solution cell mixture up and down several times to make a homogenous mixture. Collect each time point after the appropriate amount of time has passed, using the method described earlier.Incubate the samples at room temperature for five minutes to ensure the complete dissolution of RNA protein complexes. Next introduce spike-in controlled transcripts that can be detected with the methodology used. Introduce these controls into each sample at a known quantity and concentration.Using a pipette, transfer the phenylguanidine isothiocyanate solution mixture to a 2.0-milliliter microcentrifuge tube. Then add 0.2 milliliters chloroform to the phenylguanidine isothiocyanate solution mixture for every one milliliter of phenylguanidine isothiocyanate solution used. Shake the microcentrifuge tube vigorously for 15 seconds and then incubate the chloroform phenylguanidine isothiocyanate.After incubation, spin the samples at 12, 000 times g for 20 minutes at four degrees Celsius. The sample should separate into three layers. Transfer the aqueous layer to a new 2.0-milliliter microcentrifuge tube using a micropipette and be careful not to disturb the interphase during this process.Discard the interphase and organic fractions. Then add an equal volume of 2-Propanol to the aqueous fraction and invert the tube 10 times. Add up to one microliter of 20 milligram per milliliter aqueous glycogen per 20 microliters of sample, especially if the yield is expected to be low.Incubate the samples at room temperature for 10 minutes. After incubation, spin the samples. Ensure that at the end of the spin, the RNA has precipitated to form a while pellet at the bottom of the tube.With a pipette, remove and discard the supernatant, taking special care not to disturb the white RNA pellet. Add one milliliter of 75%ethanol per one milliliter of phenylguanidine isothiocyanate solution reagent to wash the pellet. Spin the samples at 12, 000 times g for 10 minutes.After removing most of the supernatant, use a pipette to remove as much ethanol as possible but do not disturb the pellet. Spins the samples to pellet all excess ethanol. The RNA pellet is less compact at this step and tends to move.After the spin, remove all excess ethanol from the tube without disturbing the pellet. Allow the RNA pellet to air-dry for five minutes. Add 50 microliters of nuclease-free water to the RNA pellet and resuspend the pellet in nuclease-free water.Treat the samples of one microliter Deanase to remove DNA and then inactivate the Deanase and cations. Store the RNA samples in 2.0-milliliter microcentrifuge tubes at minus 80 degrees Celsius. Quantify the RNA and check the RNA for purity using a spectrophotometer.After checking the purity, analyze the RNA with a 1%agarose gel to visualize the extend of degradation. Two clear bands representing 18S and 28S rRNA should be clearly visible. If these bands are not visible, the RNA may be degraded.Next, monitor the transcript abundance in the collect aliquots of RNA compared with the spiked-in internal standard using Real-Time qPCR. Microarrays, RNA sequencing, or northern blots can also be used. Fluorescence intensities over time correspond to gene expression.While fibroblasts become contact-inhibited, the transcript-encoding Collagen 3A1, a very important gene for creating the extracellular matrix deposited during wound healing becomes more stable and does not decay as rapidly. Don't forget that working with phenol can be extremely hazardous and it is important not to let it touch your skin. After watching this video, you should have a good understanding of how to monitor transcript decay rates in proliferating and quiescent fibroblasts.Changes in transcript decay rate can be an important mechanism of gene triangulation.

Research

JoVE Journal

Genetics

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Investigación

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Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

Promotor captura Hola-C: Perfiles de alta resolución, genoma de interacciones promotor

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Vigilancia de todo el genoma de errores de transcripción en organismos eucariotas

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