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.

Tutti gli animali sono stati trattati in conformità con le linee guida del National Institutes of Salute. La cura e l&#39;uso Comitato degli animali presso l&#39;Università di Alabama a Birmingham ha approvato l&#39;uso degli animali. 1. Ottenere RNA e DNA da un Arricchita astrocitica Popolazione utilizzando fluorescenti Cellulari Attivato Ordinamento (FACS) di astrociti da tutto il tessuto del cervello <ol><li> Animale sedate con CO 2 per 1 minuto e poi rapidamente decapitarlo. Sezionare cortecce come descritto in Albuquerque et al, 26.; non è necessario rimuovere meningi. Nota: i ratti transgenici che esprimono eGFP sotto il S100β (un marcatore astrociti) promotore sono stati generati da Itakura et al 27 e utilizzati per FACS ordinamento degli astrociti.. </li><li> Preparare tutto omogenato cerebrale utilizzando un kit di dissociazione papaina in condizioni non sterili, secondo il protocollo del produttore. Calore disattivare papaina a 37 ° C a bagnomaria per 10 min. Nota:Soluzione papaina è fornito dal costruttore e contiene L-cisteina e EDTA (vedi Tabella dei Materiali). <ol><li> Tagliare o dremel un foro nella parte superiore di un tubo da 50 ml per permettere tubazione che trasporta il 95% O 2: 5% CO 2 da alimentare in una provetta conica chiusa (Figura 1A). Mettere cortecce sezionato in 10 millimetri piatto di coltura contenenti supporti dissociazione (MEM supplementato con 20 mM glucosio e la penicillina / streptomicina (500 U / ml) ed equilibrato al 95% O 2: 5% di CO 2) e utilizzare una lama di rasoio pulito per tritare il tessuto in 1 x 1 mm 2 pezzi. </li></ol></li><li> Tessuto Trasferimento con 10 ml Pipetman manuale a tubo da 50 ml contenente soluzione papaina. Lasciare tessuto di depositarsi fondo pipetta prima dello scarico per minimizzare la quantità di materiale di dissociazione riportati. Mantenere soluzione papaina equilibrati a 95% O 2: 5% CO 2 attraverso lo scambio di gas di superficie per la durata dell&#39;incubazione. Non bolla papaina solution. Incubare il tessuto per 20 minuti a 37 ° C bagnomaria. </li><li> Dopo l&#39;incubazione in soluzione papaina, tessuto triturare 10 volte con una pipetta 10 ml di trasferimento a bassa velocità. Centrifugare sospensione cellulare nuvoloso a 1.000 xg per 5 minuti a temperatura ambiente. <ol><li> Equilibrare soluzione inibitore DNasi / albumina (fornito dal costruttore) attraverso lo scambio di gas superficiale e risospendere le cellule in pellet in 3 ml di soluzione inibitore / DNasi di albumina. Preparare commerciale gradiente di densità discontinuo seguendo le istruzioni del produttore. </li></ol></li><li> Spin gradiente di densità discontinuo a 1.000 xg per 6 min. Isolare le cellule dissociate dal fondo della provetta da succhiare pellet fondo con una pipetta. </li><li> Risospendere le cellule dissociate in 2-3 ml di DPBS con 0,02% di albumina di siero bovino e 1 mg / ml DNasi o HEPES buffered preferiti coltura. Passare attraverso 40 micron filtro prima FACS (Figura 2A). Mantenere le cellule in ghiaccio fino a quando l&#39;ordinamento. <ol><li> Eseguire FACS 28. &lt;/ li&gt; <li> Cellule pellet in 1,5 ml provette a 2000 xg per 5 minuti a 4 ° C. Nota: astrociti Il pellet possono essere utilizzati immediatamente o conservati in -80 ° C fino al momento dell&#39;estrazione del DNA. </li></ol></li><li> Estratto RNA e DNA utilizzando il metodo preferito di isolamento 29 30. Valutare RNA e concentrazione del DNA e la qualità con spettrofotometro e bioanalizzatore 31. Nota: Utilizzare solo RNA alta qualità e il DNA in fasi successive, 260/280 = 2,0-2,2 e 1,8-1,9, rispettivamente. Analisi Bioanalyzer è essenziale per valutare la degradazione dell&#39;RNA o frammentazione del DNA. RNA o DNA possono essere utilizzati immediatamente o conservate in -80 ° C o -20 ° C, rispettivamente per gli studi successivi. </li></ol> 2. Valutare metilazione del DNA Stato di un gene mediante metilazione-sensibile ad alta risoluzione Melt Analysis (MS-HRMA) <ol><li> Inserisci sequenza del gene di interesse in preferito software di mappatura metilazione online per individuare eventuali isole CpG del genedi interessi 32. <ol><li> Primer design contro bisolfito convertiti sequenza di DNA 33 e amplificano con DNA polimerasi preferita secondo il protocollo del produttore. Verificare le dimensioni del prodotto amplificato mediante l&#39;esecuzione di un gel di DNA agarosio 1% a 100 V per 45 minuti. Conservare primer a concentrazione magazzino di 20 micron di -20 ° C. </li></ol></li><li> Bisolfito convertire 500-1000 ng di DNA di ciascun campione e DNA metilato standard che vanno da 0-100% delle stesse specie animali 34. Campioni eluire per fornire una concentrazione di 20 ng / ml. Verificare la concentrazione di bisolfito di DNA convertito con spettrofotometro 31. </li><li> Setup 20 reazioni microlitri per l&#39;amplificazione di MS-HRM utilizzando DNA polimerasi preferito e primer a 5 micron di concentrazione in base alla tabella 1 e 2. Eseguire tutti i campioni, tra cui DNA e metilato standard FACS, in triplice copia. </li><li> A seconda del software di analisi, impostare pre- e post-avviare e arrestare i parametriintorno alle transizioni della curva di fusione. Set inizio pre-fusione e parametri di stop pre-melt così la differenza tra i due è 0,2-0,5 ° C. Imposta l&#39;inizio post-fusione e post-fusione fermare in modo simile. Estrarre i dati differenza di temperatura di picco per ogni campione. </li><li> Utilizzando gli standard per cento metilati (y-valore) e le loro corrispondenti differenze di temperatura di picco medi (x valore) genera una equazione di regressione lineare (Figura 3A-B, Tabella 3-4). Utilizzare questa equazione di regressione lineare per stimare lo stato di metilazione di 35 campioni sconosciuti. </li></ol> 3. Valutare Hyper-metilato Promotore Attività via Uso di luciferasi Assay <ol><li> Identificare isole CpG del gene bersaglio (punto 2.1). PCR amplifica regioni di interesse 36 e clone monte della luc2 Firefly gene della luciferasi giornalista per la produzione di plasmidi CpG-isola-luc2 37. </li><li> Linearizzare 30 microgrammi di plasmide CpG-isola-luc2 via restrizionedigestione enzimatica 38. Verificare i siti di restrizione digest e di evitare doppi tagli utilizzando il software di taglio preferito 38. Calore-inattivare enzimi a temperatura e durata adeguata seguenti digestione secondo il protocollo del produttore. Perdita minima di DNA si verifica durante la fase di linearizzazione. </li><li> Metilato plasmidi linearizzati con CpG metilasi (M.Sssl) O / N a 30 ° C o lasciare non trattati protocollo seguente produttore tranne che per le seguenti regolazioni. </li><li> Eseguire 50 reazioni microlitri. </li><li> Utilizzare 5 unità di CpG metilasi per metilare 700 ng di plasmide linearizzato. </li><li> Eseguire reazioni O / N per 13-19 ore. </li><li> A seguito di CpG reazione metilasi, eseguire la pulitura del DNA utilizzando lo standard, disponibile in commercio membrana di gel di silice pulizia Kit secondo il protocollo del produttore. Perdite significative di DNA si verificano a seguito CpG reazione metilasi, perdita 30-60%. </li><li> Verificare metilazione di plasmidi tramite una restrizione Hpa II digest. </li&gt; <li> Prendere 1 mg di DNA metilato o non metilato e restrizione digerire con Hpa II per 1 ora a 37 ° C. Utilizzare 5-10 unità di Hpa II per ogni mg di DNA. A seguito di Hpa II digestione, eseguire la pulitura DNA usando preferita, membrana disponibile in commercio gel di silice ripulire kit. Eseguire entrambi plasmidi metilati e non metilati CpG su un gel di agarosio 1% DNA in tampone TAE o TBE a 100 V per 45 minuti per la visualizzazione (Figura 4B). </li><li> Oggetto sia denaturato e non metilato plasmidi a doppia digestione con opportuni enzimi di restrizione per rilasciare full-length luc 2 (vettoriale) e l&#39;isola CpG (inserto) frammenti 38. Eseguire doppio digestione O / N a temperatura adeguata e gli enzimi calore inattivare seguente digestione secondo il protocollo del produttore. Minima perdita di concentrazione di DNA avviene durante doppia restrizione digest. </li><li> Eseguire plasmidi doppio digeriti su 1% gel di agarosio DNA a 100 V per 1 ora per permettere la separazione of vettore e inserire (Figura 4C). Attenzione. Indossando UV scudo protettivo faccia, mettere il gel del DNA su un tavolo nero luce di visualizzare bande. In base alle dimensioni, accise inserto metilato e non metilato e non metilato vettore con una lama chirurgica pulito. </li><li> Gel estratto di DNA con fenolo saturo, pH 6,6. In breve, pesare gel DNA contenente vettoriale o inserto. Utilizzare 100 ml di fenolo per 0,1 grammi di gel DNA. Omogeneizzare gel DNA fenolo utilizzando Dounce vetro omogeneizzatore. </li><li> Aggiungere cloroformio (con 1/5 del volume di fenolo) ed agitare i campioni per 20 sec. Incubare i campioni a temperatura ambiente per 2-3 minuti. Centrifugare per 15 minuti a velocità massima (16,1 x 1.000 xg) a 4 ° C. </li><li> Rimuovere la soluzione acquosa e 0,1 X. aggiungere volume di 3 M acetato di sodio e 2,5 volte il volume di etanolo. Incubare i campioni per 1 ora a -80 ° C. Dopo l&#39;incubazione, rimuovere l&#39;etanolo e risospendere il DNA in 30 microlitri di tampone preferito. Significativa perdita di DNA avviene seguenti isolamento di inserti e vettori, 30-50% loss. </li><li> Re-legare inserti metilato e non metilato al vettore non metilato utilizzando T4 DNA ligasi 38. Utilizzare un rapporto 1: 4 di vettore da inserire per le reazioni legatura. Reazione di installazione in base alle istruzioni del produttore. Utilizzare un volume totale di 50 microlitri per le reazioni e incubare O / N a -20 ° C o su ghiaccio con coperchio sopra secchiello del ghiaccio. </li><li> A seguito di legatura, eseguire la pulitura DNA usando preferita, membrana disponibile in commercio gel di silice ripulire kit. Significativa perdita di DNA si verificano a seguito di reazione legatura, un approssimativo perdita 30-50% del DNA. Verificare ri-legatura eseguendo il 1% gel di DNA agarosio a 100 V per 45 minuti e valutare la concentrazione di plasmidi ri-legatura (Figura 4D). </li><li> Trasfettare plasmidi denaturato o non denaturato nelle cellule D54 con un reagente di trasfezione commerciale secondo il protocollo del produttore. Cellule D54 Seed sulla piastra 12 pozzetti alla 0,14 x 10 6 cellule / pozzetto. </li><li> A seguito di 24 ore, le cellule trasfettare with concentrazioni uguali di entrambi (1) plasmide non methyated CpG luc2 + Renilla o un altro controllo luciferasi vettoriale o (2) metilata plasmide CpG luc2 + Renilla o un altro controllo luciferasi vettoriale. </li><li> Consentono alle cellule per trasfettare per 24 ore prima di eseguire doppio test luciferasi. Eseguire test secondo le istruzioni del produttore. Eseguire le letture usando un luminometro. Leggere ogni bene in triplice copia. </li><li> Calcolare il rapporto di attività luciferasi Firefly a Renilla o altre attività di controllo della luciferasi. Normalizzare metilato Firefly: attività luciferasi di controllo per non metilato Firefly: attività luciferasi controllo dividendo l&#39;attività luciferasi metilato per attività luciferasi non denaturato </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 popolazione arricchita di astrociti è stato acquisito tramite FACS ordinamento di eGFP-S100β animali transgenici 27. A causa della diminuzione della qualità delle cellule e molecole molecolari isolati da animali di età superiore a postnatale giorno 50 (p50), animali di età p0-P40 sono ottimali per tali esperimenti. Tessuto corticale è stato utilizzato per questi esperimenti. Cortecce da due a sei animali sono stati riuniti insieme. FACS è stata eseguita presso lo stabilimento UAB Comprehensive Cito...

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.

Correlare DNA Modifiche metilazione gene-specifica con espressione e trascrizionale attività di astrociti<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 Views.  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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Ricerca

Biologia

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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.

Determinazione della metilazione del DNA di geni impressa Arabidopsis Dell&#39;endosperma

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.

Metilazione del DNA: Modifica bisolfito e analisi

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.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

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 quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

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 metilazione Assay per studiare proteine ​​Arginina Metilazione

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 ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

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.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

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 ...