Questo lavoro è stato sostenuto dalle concessioni dal National Institute of Health RO1-GM085130 e la National Science Foundation MCB0929394. Ringraziamo il Dott. Marion Schmidt per la sua ospitalità e per averci permesso di filmare nel suo laboratorio.

1. Preparazione dei reagenti Footprinting <ol><li> Preparare un tampone di reazione 10x contenente 100 cacodilato sodio mM, 1 mM EDTA, e 1 M KCl. Regolare il pH a 7,4. Filtrare il tampone usando un dispositivo di acetato 0,2 mM filtro (Nalgene). Nota: non RNA pipettare direttamente nel buffer 10x. </li><li> Preparare la miscela di reazione di titolazione per ogni reazione, come indicato nella tabella 1. Il volume del mix di titolazione (1x buffer e Mg (II) alla concentrazione desiderata) dovrebbe essere di 90 microlitri, prima di aggiungere 10μl di RNA nel buffer 1x. </li><li> Preparare una digestione RNasi T1 tampone contenente urea 6.63M, citrato di sodio 20 mM, 1mM EDTA, 0,25 mg / tRNA microlitri, cyanol xilene 0,025%, e il blu bromofenolo 0,025%. Questo buffer può essere conservato a 4 ° C per un massimo di 6 mesi. </li><li> La reazione di Fenton richiede preparati al momento le soluzioni acquose di 250 mm Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA, sodio e 500 mM L-ascorbato. Le concentrazioni osservato nel capitolo 10,5 sono ottimali per esperimenti di equilibrio in un tampone come cacodilato di sodio che non eliminano i radicali e diverse concentrazioni di ioni bivalenti. </li><li> Reagenti per la perossidazione (1.5.1) e di ossidazione (1.5.2) di reazione footprinting. <ol><li> La reazione di Fenton perossidativi richiede preparati al momento le soluzioni acquose di 100 mM Fe-EDTA soluzione di sodio e 50 mM di L-ascorbato e 1,5% di H 2 O 2. La Fe-EDTA soluzione viene preparata mescolando Fe (NH 4) 2 (SO 4) 2 con EDTA di avere 1,1 volte l&#39;eccesso di EDTA su ferro. </li><li> Poco prima di iniziare la reazione ossidativa scissione (3.4.2), creare il mix reazione di Fenton, combinando 2 l di Fe (NH 4) 2 (SO 4) 2 (250 mm), 2,2 ml di EDTA (250 mm), 62,5 microlitri L-ascorbato di sodio (500 mm), 22,5 reazione microlitri di buffer 10x e 135,5 ml di H 2 O per raggiungere le concentrazioni finali nella miscela di reazione footprinting (3.4.2) pari a 0.10 mM, 0,11 mm e 6,61 mm, rispettivamente. </li></ol></li></ol> 2. Preparazione di RNA per Esperimento Footprinting <ol><li> L&#39;RNA può essere prodotto da standard di trascrizione del DNA in vitro di modelli 6 o, se più corta di 50 nucleotidi, essere acquistato (ad es Integrated Technologies DNA, www.idtdna.com). Seguire le procedure di pulizia raccomandato di laboratorio per mantenere l&#39;integrità del campione di RNA durante gli esperimenti footprinting 7. </li><li> Defosforilazione 8 di RNA trascritto in vitro è necessario prima di 32 etichettatura P del 5 &#39;di RNA. 10 pmol di RNA sono end-etichettati da kinasing. 9 La soluzione deve essere torbida dopo il successo inattivazione della fosfatasi. </li><li> Purificare l&#39;RNA radioattivo mediante elettroforesi su gel denaturante. L&#39;RNA viene recuperato da due successive estrazioni gel utilizzando acetato di sodio 0,3 M (pH 5,5). RNA è precipitato mescolando 0,5 ml di soluzione acquosa con 1 ml di etanolo e successiveincubazione per 1 minuto in ghiaccio secco. Spin campione a 4 ° C, 12500 rpm per 0,5 ore. Eliminare il surnatante e lavare RNA a pellet con etanolo al 70%. Rimuovere il surnatante dopo centrifugazione ulteriore e secco di pellet nel vuoto. </li><li> Sciogliere il 32 P-etichettati campioni di RNA in 330 ul di 1x Buffer.Pipet 30 microlitri della soluzione di RNA in un tubo di reazione, precipitato con etanolo e l&#39;RNA secco nel vuoto; tenere questo tubo per generare la scala di riferimento (punto 3.5). Denaturare la soluzione tamponata di RNA da riscaldamento a 95 ° C per 2 min. Raffreddare i campioni a temperatura ambiente per 15 minuti e dare un giro rapido per abbattere i condensati sui coperchi dei tubi di riportare in soluzione. </li></ol> 3. Il Footprinting Experiment <ol><li> Il pettine elettroforesi determina il numero massimo di punti dati per gel analitici. Noi usiamo un 30 pettine bene. La scala di riferimento occupa 2 pozzi e quello di controllo uncleaved. Preparare 27 provette di reazione. La finale Mg (II) Concentration deve essere equamente distanziati su una scala logaritmica si estende ordini di grandezza attorno al punto medio di titolazione. </li><li> Separatamente, incubare l&#39;RNA e la (II) soluzioni Mg a 50 ° C per 5 min. Miscelare 10 ml di soluzione di RNA con 90 microlitri della corrispondente quantità di Mg (II) in 1x tampone per raggiungere un volume finale di 100 ul. Incubare le soluzioni per 30 minuti a 50 ° C. </li><li> Lasciate che le soluzioni equilibrare a 25 ° C per 1 ora, mentre si piega. </li><li> Perossidativi (3.4.1) o ossidativi (3.4.2) idrossile reazione footprinting radicale. <ol><li> Avviare la reazione di perossidazione mettendo gocce di 2 microlitri Fe-EDTA (100 mm), 2 microlitri di sodio L-ascorbato (50 mm) e 2 microlitri H 2 O 2 (1,5%), separati l&#39;uno dall&#39;altro nella parte superiore interna della reazione tubo contenente la soluzione di RNA e avviare la reazione di footprinting mescolando vigoroso. Arrestare la reazione dopo 15 secondi con l&#39;aggiunta di 300 ml di etanolo assoluto freddo. Girare i tubi 3-5 volte. Precipitato, lavaggio easciugare il pellet come al punto 2.4. </li><li> Avviare il ossidativo footprintingreaction radicale ossidrile aggiungendo 5 ml di preparati al momento della miscela di reazione Fenton (1,6). Incubare per 30 min a 25 ° C. Aggiungere 300 ml di etanolo assoluto freddo per placare la reazione e mescolare girando i tubi 3-5 volte. Precipitato, lavare e asciugare il pellet come indicato al punto 2.4. </li></ol></li><li> Generare un RNasi T1 campione di riferimento digerire secondo le procedure standard 10 utilizzando la soluzione preparata al punto 1.3. </li><li> Sciogliere pellet di RNA a 8 microlitri gel colorante carico II (Ambion) e confermare l&#39;RNA viene risospeso utilizzando il contatore Geiger. </li><li> Preparare una denaturazione, 8% gel di sequenziamento poliacrilammide secondo protocolli standard. 11 campioni di carico tra cui due riferimenti e un controllo. Separare frammenti di RNA a 60 W - 75 W per 2,5 ore. Esporre gel essiccato ad uno schermo ai fosfori durante la notte. Scansione schermo al fosforo con un sistema di imaging per autoradiografia filmless, ad esempio,. Tempesta 865 (GE Healthcare) o Typhoon (GE Healthcare). Trasferimento di file immagine di gel sul tuo computer. </li></ol> 4. Analisi dei dati <ol><li> La reattività del radicale idrossile ogni nucleotide è determinato da quantificare l&#39;intensità di ogni banda sul gel analitici. Scaricare, installare e aprire SAFA, un facile da usare software open source per il montaggio singola banda e la quantificazione. 12, 13, seguire le istruzioni per l&#39;uso manuale. In breve, caricare la sequenza di RNA come file. Txt seguito dalla foto come gel. Gel file. Regolare intensità di banda, definire i vicoli, scegliere una corsia di ancoraggio, ed eseguire un allineamento gel. Assegnazione di bande di nucleotidi si verifica in riferimento alla scala RNasi T1 digestione. Quantificare gli integrali banda e utilizzare la normalizzazione / colorplot funzione per normalizzare e assegnare i residui potenzialmente invarianti. Salvare l&#39;output come file. Txt. </li><li> SAFA uscite un foglio di calcolo contenente le colonne che rappresentano corsie sul gel e le righe che rappresentano i integrated densità banda delle bande isolata corrispondente al frammento di RNA. In primo luogo, individuare i siti della protezione che mostrano un notevole cambiamento nella accessibilità al solvente confrontando il profilo di protezione deriva dalla non Mg (II) campione con il profilo del punto finale Mg (II) concentrazione (50 mM nel nostro esempio). Più basso è il valore, più protetto il nucleotide è contro l&#39;attacco dei radicali idrossili e viceversa. </li><li> Crea isoterme pieghevole dal integrali banda normalizzata di singoli o di gruppi di nucleotidi contro Mg (II) la concentrazione. Le isoterme sono individualmente scalata a saturazione frazionale di fi = L + (U - L) • <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> dove f indica la densità integrata della band (s) oggetto di analisi, L e U rappresentano i limiti inferiore e superiore alla transizione e <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> è la saturazione frazionale. 14 I dati sono idonei con una non-lineare lest piazza programma di analisi (si può usare o Origin (OriginLab) o GraphPad (GraphPad Software, Inc.).) all&#39;equazione Hill (1), <img alt="Equazione 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> dove K d è l&#39;equilibrio costante di dissociazione, [M] corrisponde alla concentrazione della variabile che media la reazione pieghevole, Mg (II) in questo esempio, e n H è il coefficiente di Hill. Questo scala procedura la transizione alla saturazione frazionale, determina il suo punto medio e fornisce una prova fenomenologico di se una transizione è sigmoidale. Isoterme derivati ​​dal sequenziamento gel (Figura 3A) che sono stati analizzati da SAFA (Figura 3B) e in forma, come descritto sopra sono mostrati in Figura 3C. Le isoterme di saturazione frazionale mostrato in Figura 3 sono stati generati dalla scala in un foglio i valori generati dalla SAFA migliori contro i limiti in forma superiore e inferiore (U e L), utilizzando la frazione equazione = valore / (U - L) - L / (U - L). </li></ol> 5. Rappresentante dei risultati: La figura 3 mostra i risultati rappresentante P4-P6 RNA • esperimenti footprinting OH. L&#39;immagine gel (a sinistra) indica che a) scissione di fondo è minima (corsia di destra), b) l&#39;assegnazione singolo nucleotide è possibile grazie alle bande ben definite nella corsia T1 (RNasi T1 si unirà ad ogni G), ec) il radicale ossidrile frammentazione indotta di RNA è ben al di sopra di fondo. Il passaggio da bassa ad alta Mg (II) è affiliato con la diminuzione della densità integrata banda di singoli e gruppi di bande indicando formazione della struttura terziaria dell&#39;RNA. Singoli o gruppi di nucleotidi contigui la cui scissione cambiamenti in concomitanza sono indicati come una &#39;protezione&#39;. Il • Protezioni OH caratteristica del Mg (II) mediata ripiegamento della P4-P6 RNA strettamente corrispondere alle regioni solvente inaccessibili della molecola osservata nella sua struttura cristallina. 15 Molecole di RNA ripiegato c<html> uno hanno estensioni molto diverse (ad esempio, quanto vicino al fondo del declino banda densità) di protezione dei contatti terziario che non sono stati correlati con evidente fenomeno strutturale e dinamico. Così, alcune molecole di RNA mostrerà ben definite transizioni strutturali, come mostrato in Fig. 3, e altri no. Intensità di banda sono quantificati e normalizzati per SAFA 12 analisi (Figura 3B). L&#39;uscita è un complotto &#39;termico&#39; visualizzando il relativo grado di protezione contro • OH. La transizione dei colori descrive il cambiamento di accessibilità su Mg (II) aggiunta. Bianco a spettacoli rosso o blu nucleotidi più accessibile o più protetti, rispettivamente. Ogni grado di ombreggiatura è affiliato con un valore numerico che può essere tracciata una curva di protezione (Figura 3C) e analizzati da un modello di associazione come ad esempio l&#39;equazione di Hill (1). La costante di equilibrio di dissociazione affiliato Protezione 153-155 è all&#39;incirca doppio rispetto a quello del corrispondente valore di Protezione 163-164. CONTENUTO &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Figura 1. Produzione di radicali ossidrile e P4-P6 pieghevole RNA. A) Fe (II) catalizza la produzione di radicali idrossili da perossido di idrogeno. Ascorbato riduce Fe (III) torna a ferro ferroso. B) In assenza di Mg (II), non P4-P6 struttura terziaria è formato, permettendo di penetrare radicali idrossili e unirà tutte le posizioni spina dorsale accessibile. Aggiunta di Mg (II) avvia pieghevole del P4-P6, consentendo solo la spina dorsale solvente accessibile ad essere spaccati dai radicali idrossili. <img alt="Figura 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Figura 2. Schema di esperimento di footprinting radicali idrossile. A) Dephophorylation e 32 P 5&#39;-end di etichettatura di RNA. B) Purificazione di 32 P-RNA marcato su un gel di poliacrilammide denaturante. C) Escissione di banda di RNA, dopo l&#39;estrazione di RNA, e la precipitazione in etanolo. D) Prefolding epieghevole di RNA. E) L&#39;aggiunta di preparati al momento miscela di reazione Fenton per generare i radicali idrossili. F) separazione frammento di RNA con elettroforesi su gel di poliacrilammide denaturante. G) La determinazione quantitativa di frammenti di RNA da un software SAFA. <img alt="Figura 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Figura 3. Analisi dei frammenti di RNA dopo perossidativi reazione footprinting Fenton. (A) L&#39;RNA è stato esposto ai radicali idrossile e prodotti di dissociazione sono stati risolti mediante elettroforesi su gel denaturante di poliacrilamide (PAGE). L&#39;immagine mostra i prodotti di perossidazione scissione con crescente concentrazione di Mg (II). Corsie di riferimento e di controllo sono etichettati. Questo file immagine è l&#39;ingresso per il programma SAFA che quantitates il volume di ciascuna banda. (B) La trama &#39;termica&#39; generato da SAFA. (C) Un foglio di calcolo dei valori relativi densità integrata banda all&#39;uscita numero nucleotide da SAFA è analizzato da un modello di associazione come la equati Hillsu (1). Aree di protezione sono stati scelti in base all&#39;aumento della densità di banda integrale in funzione di Mg (II) la concentrazione. D K è la (II), Mg concentrazione alla quale è piegato metà del RNA nel sito monitorato. Il coefficiente di Hill, n H, è una misura della pendenza della curva che fornisce informazioni sulla cooperatività vincolante e fornisce una stima più bassa per il numero di ioni magnesio coinvolti nel ripiegamento di questo particolare sito. <img alt="Tabella 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tabella 1. Volumi Rappresentante per la generazione di campioni di RNA contenenti differenti Mg (II) le concentrazioni.

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Nessun conflitto di interessi dichiarati.

Footprinting radicale ossidrile è uno strumento prezioso per valutare l&#39;area solvente superficie accessibile di acidi nucleici. Formazione qualitativa e quantitativa della struttura terziaria 14 può essere seguita in funzione di parametri quali il tipo e la concentrazione di ioni, pH, temperatura, proteine ​​di legame o pieghevoli co-fattori. La combinazione vincente di un protocollo di dritto in avanti ed economico e l&#39;accessibilità conseguente solvente e le informazioni su un pieghevole a sin...

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	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

Molecole di RNA svolgono un ruolo essenziale in biologia. Oltre alla trasmissione di informazioni genetiche, l&#39;RNA può piegare in unico strutture terziarie che compie un ruolo biologico specifico come regolatore, legante o catalizzatore. Informazioni sulla formazione terziaria contatto è essenziale per capire la funzione di molecole di RNA. I radicali idrossile (OH •) sono le sonde della struttura degli acidi nucleici a causa della loro elevata reattività e piccole dimensioni. 1 Quando utilizzato come sonda footprinting, i radicali idrossili mappare la superficie solvente accessibile della dorsale fosfodiestereo di DNA 1 e 2 con RNA sottile come singolo nucleotide risoluzione. Idrossile footprinting radicale può essere utilizzato per identificare i nucleotidi all&#39;interno di una superficie di contatto intermolecolari, ad esempio nel DNA-proteina 1 e RNA-proteina complessi. Equilibrio 3 e 4 transizioni cinetica può essere determinato da condurre footprinting radicale ossidrile in funzione di una soluzione moltoil variabile o il tempo, rispettivamente. Una caratteristica fondamentale di footprinting è che l&#39;esposizione limitata alla sonda (per esempio, &#39;single-hit cinetica&#39;) i risultati nel campionamento uniforme di ogni nucleotide del polimero 5. In questo articolo il video, si usa il P4-P6 dominio del ribozima Tetrahymena per illustrare la preparazione dei campioni di RNA e la determinazione di un (II)-mediata isoterme Mg pieghevole. Descriviamo l&#39;uso del noto protocollo di idrossile footprinting radicale che richiede H 2 O 2 (chiamiamo questo il protocollo &#39;perossidativi&#39;) e un valore, ma poco noto, alternativa che utilizza naturalmente disciolto O 2 (lo chiamiamo la &#39; ossidativo &#39;protocollo). Una panoramica della riduzione dei dati, la trasformazione e procedure di analisi è presentato.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

Questo protocollo descrive come quantificare il Mg (II)-dipendente formazione della struttura terziaria dell&#39;RNA con due metodi di footprinting radicali idrossili.

Cambiamenti di equilibrio nella struttura di monitoraggio RNA da &#39;perossidativi&#39; e &#39;ossidativo&#39; Footprinting radicale ossidrile

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K Views.  Hunter College. Questo protocollo descrive come quantificare il Mg (II)-dipendente formazione della struttura terziaria dell'RNA con due metodi di footprinting radicali idrossili.

Video: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Metodi di singola molecola per monitorare l&#39;evoluzione a doppio strato: Proprietà meccaniche

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Ricerca

Biologia

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Rilevamento delle modifiche droga indotte recettore post-internalizzazione Tratta di Analisi Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...