Ce travail a été soutenu par des subventions du National Institute of Health RO1-GM085130 et la National Science Foundation MCB0929394. Nous remercions le Dr Marion Schmidt pour son hospitalité et pour nous permettre de filmer dans son laboratoire.

1. Préparation des réactifs Footprinting <ol><li> Préparer un tampon de réaction 10x contenant 100 mM cacodylate de sodium, EDTA 1 mM, et 1 M KCl. Ajuster le pH à 7,4. Filtrer le tampon en utilisant un dispositif de filtre acétate de 0,2 pM (Nalgene). Remarque: ne pas ARN pipette directement dans la mémoire tampon 10x. </li><li> Préparer le mélange réactif de titrage pour chaque réaction, comme indiqué dans le tableau 1. Le volume du mélange de titration (1x tampon et Mg (II) à la concentration souhaitée) doit être de 90 ul, avant d&#39;ajouter 10 ul d&#39;ARN dans un tampon de 1x. </li><li> Préparer un tampon de digestion RNAse T1 contenant de l&#39;urée 6.63M, citrate de sodium 20 mM, 1 mM EDTA, 0,25 pg / ul ARNt, cyanol xylène 0,025%, et le bleu de bromophénol 0,025%. Ce tampon peut être conservé à 4 ° C pendant jusqu&#39;à 6 mois. </li><li> La réaction de Fenton nécessite fraîchement préparés solutions aqueuses de 250 mM de Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA et 500 mM de sodium L-ascorbate. Les concentrations indiquées dans le chapitre 10.5 sont optimales pour des expériences de l&#39;équilibre dans un tampon de cacodylate de sodium, comme cela ne veut pas piéger les radicaux et divers concentrations en ions divalents. </li><li> Réactifs pour la réaction empreinte (1.5.2) peroxydante (1.5.1) et oxydante. <ol><li> La réaction de Fenton peroxydante nécessite fraîchement préparés solutions aqueuses de 100 mM de Fe-EDTA et 50 mM de sodium L-ascorbate et de 1,5% H 2 O 2. La solution Fe-EDTA est préparée en mélangeant Fe (NH 4) 2 (SO 4) 2 avec de l&#39;EDTA à avoir 1,1 fois plus de l&#39;EDTA sur le fer. </li><li> Juste avant d&#39;initier la réaction de clivage oxydatif (3.4.2), de créer le mélange réaction de Fenton en combinant 2 pi de Fe (NH 4) 2 (SO 4) 2 (250 mm), 2,2 l de EDTA (250 mM), 62,5 ul L-ascorbate de sodium (500 mM), 22,5 pi de tampon de réaction 10x et 135,5 ul de H 2 O pour atteindre les concentrations finales dans le mélange réactionnel empreinte (3.4.2) de 0.10 mM, 0,11 mM et 6,61 mM, respectivement. </li></ol></li></ol> 2. Préparation de l&#39;ARN pour l&#39;expérience d&#39;empreintes <ol><li> L&#39;ARN peut être produite par la norme de transcription in vitro de 6 modèles de l&#39;ADN ou, si courte que 50 nucléotides, être achetés (par exemple chez Integrated DNA Technologies, www.idtdna.com). Suivez les procédures recommandées pour la propreté de laboratoire afin de maintenir l&#39;intégrité des échantillons d&#39;ARN au cours des expériences empreintes 7. </li><li> La déphosphorylation 8 du transcrit in vitro d&#39;ARN est nécessaire avant d&#39;étiquetage P 32 de l&#39;extrémité 5 &#39;de l&#39;ARN. 10 pmol d&#39;ARN sont marqués aux extrémités par des kinasing 9. La solution devrait être nuageux après inactivation de la phosphatase succès. </li><li> Purifier l&#39;ARN radiomarqué en utilisant l&#39;électrophorèse sur gel dénaturant. L&#39;ARN est récupéré par deux extractions ultérieures gel en utilisant l&#39;acétate de sodium 0,3 M (pH 5,5). L&#39;ARN est précipité en mélangeant 0,5 ml de solution aqueuse à 1 ml d&#39;éthanol et ultérieuresd&#39;incubation de 1 minute sur la glace sèche. Spin échantillon à 4 ° C, 12 500 rpm pendant 0,5 heure. Jeter le surnageant et laver avec de l&#39;éthanol culot d&#39;ARN de 70%. Retirer le surnageant après centrifugation supplémentaire et culot sec dans le vide. </li><li> Dissoudre le 32 P-étiqueté les échantillons d&#39;ARN dans 330 ul de 1x Buffer.Pipet 30 pl de la solution de l&#39;ARN dans un tube de réaction, un précipité avec l&#39;éthanol et de l&#39;ARN à sec dans le vide; garder ce tube pour générer l&#39;échelle de référence (étape 3.5). Dénaturer la solution tamponnée ARN par chauffage à 95 ° C pendant 2 min. Refroidir les échantillons à température ambiante pendant 15 minutes et donner un tour rapide pour faire baisser les condensats sur les couvercles des tubes dans la solution. </li></ol> 3. L&#39;expérience d&#39;empreintes <ol><li> Le peigne électrophorèse détermine le nombre maximal de points de données par gel analytique. Nous utilisons un peigne à 30 puits. L&#39;échelle de référence occupe 2 puits et le contrôle non clivée. Préparer 27 tubes de réaction. La finale Mg (II) concentrations doivent être espacés uniformément sur une échelle logarithmique couvrant plusieurs ordres de grandeur autour du point médian de titrage. </li><li> Par ailleurs, incuber l&#39;ARN et le Mg (II) des solutions à 50 ° C pendant 5 min. Mélanger 10 ul de la solution d&#39;ARN avec 90 ul de la quantité correspondante de Mg (II) en 1x tampon pour atteindre un volume final de 100 pl. Incuber les solutions pendant 30 min à 50 ° C. </li><li> Laissez les solutions s&#39;équilibrer à 25 ° C pendant 1 heure tout en pliage se produit. </li><li> Peroxydante (3.4.1) ou oxydatif (3.4.2) hydroxyle réaction empreinte radicale. <ol><li> Démarrer la réaction peroxydante en plaçant des gouttelettes de 2 pl Fe-EDTA (100 mm), 2 ul de sodium L-ascorbate (50 mm) et 2 ul H 2 O 2 (1,5%), séparés les uns des autres à l&#39;intérieur haut de la réaction tube contenant la solution d&#39;ARN et de démarrer la réaction empreinte en mélangeant vigoureusement. Arrêter la réaction après 15 secondes en ajoutant 300 ul d&#39;éthanol froid absolu. Tournez les tubes 3-5 fois. Précipité, lavez etsèche le culot comme à l&#39;étape 2.4. </li><li> Démarrez le oxydatif footprintingreaction radical hydroxyle en ajoutant 5 ul du mélange de réaction de Fenton fraîchement préparés (1,6). Incuber pendant 30 min à 25 ° C. Ajouter 300 ul d&#39;éthanol absolu froid pour stopper la réaction et mélanger en tournant les tubes 3-5 fois. Précipité, laver et sécher le culot, comme indiqué dans l&#39;étape 2.4. </li></ol></li><li> Générer un échantillon de référence RNase T1 digérer, conformément aux procédures standard de 10 en utilisant la solution préparée à l&#39;étape 1.3. </li><li> Dissoudre culots d&#39;ARN dans les 8 ul du gel de chargement II colorant (Ambion) et confirmer l&#39;ARN est remis en suspension par l&#39;aide du compteur Geiger. </li><li> Préparer une dénaturation, un gel de polyacrylamide 8% séquençage selon des protocoles standard. 11 Charger les échantillons dont deux références et un contrôle. Séparer des fragments d&#39;ARN à 60 W - 75 W pendant 2,5 heures. Exposer gel séché sur un écran de phosphore de stockage pendant la nuit. Balayage écran phosphorique avec un système d&#39;imagerie pour autoradiographie sans film, par exemple,. Tempête 865 (GE Healthcare) ou Typhoon (GE Healthcare). Transfert de fichier image du gel sur votre ordinateur. </li></ol> 4. Analyse des données <ol><li> La réactivité du radical hydroxyle de chaque nucléotide est déterminé par la quantification de l&#39;intensité de chaque bande sur le gel analytique. Téléchargez, installez et ouvert SAFA, un outil facile à utiliser un logiciel open source pour le montage et la quantification seule bande. 12, 13 Suivez les instructions du manuel utilisateur. Brièvement, la charge de la séquence d&#39;ARN sous. Txt suivie par l&#39;image gel comme. Gel de fichier. Ajustez intensités des bandes, de définir les voies, choisissez une voie d&#39;ancrage, et d&#39;effectuer un alignement de gel. Attribution des bandes de nucléotides se produit en référence à l&#39;échelle de digestion RNase T1. Quantifier les intégrales bande et utiliser la fonction de normalisation / colorplot à normaliser et à attribuer des résidus potentiellement invariant. Enregistrez le résultat dans un fichier. Txt. </li><li> SAFA sorties d&#39;un tableur contenant des colonnes représentant voies sur le gel et des lignes représentant l&#39;enINTÉGRÉ densité de la bande des bandes individuelles correspondant au fragment d&#39;ARN. Tout d&#39;abord, identifier les sites de protection qui affichent un changement notable dans l&#39;accessibilité de solvant en comparant le profil de protection dérivée de la non Mg (II) de l&#39;échantillon avec le profil du point de fin de Mg (II) de concentration (50 mM dans notre exemple). La baisse de la valeur, le plus protégé le nucléotide est contre l&#39;attaque des radicaux hydroxyles et vice versa. </li><li> Créer isothermes pliage de la bande normalisée de intégrales ou des groupes de nucléotides par rapport Mg (II) de concentration. Les isothermes sont individuellement mis à l&#39;échelle de la saturation fractionnelle par fi = L + (U - L) • <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> où f représente la densité intégrée de la bande (s) en cours d&#39;analyse, L et U représentent les limites inférieures et supérieures à la transition et <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> est la saturation fractionnelle. 14 Les données sont adaptées en utilisant un L non-linéaireprogramme d&#39;analyse de l&#39;Est carrés (Nous utilisons soit l&#39;origine (OriginLab) ou GraphPad (GraphPad Software, Inc.)) à l&#39;équation de Hill (1), <img alt="L&#39;équation 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> où K d est la constante de dissociation d&#39;équilibre, [M] correspond à la concentration de la variable qui intervient dans la réaction de repliement, Mg (II) dans cet exemple, et n H est le coefficient de Hill. Cette procédure d&#39;échelles de la transition vers la saturation fractionnelle, détermine son milieu et fournit un test phénoménologique de savoir si une transition est sigmoïde. Isothermes dérivées de gels de séquençage (figure 3A) qui ont été analysés par la SAFA (figure 3B) et ajustement tel que décrit ci-dessus sont présentés dans la figure 3C. Les isothermes de saturation fractionnelle montre la figure 3 ont été générés par l&#39;échelonnement dans un tableur les valeurs générées par la SAFA contre le meilleur ajustement des limites supérieure et inférieure (U et L) en utilisant la fraction équation de valeur = / (U - L) - L / (U - L). </li></ol> 5. Les résultats représentatifs: La figure 3 montre des résultats représentatifs de P4 à P6 ARN • expériences empreinte OH. L&#39;image du gel (à gauche) indique que le clivage de fond) est minime (voie de droite), b) l&#39;affectation simple nucléotide est possible grâce à des bandes bien définies dans la voie de T1 (T1 RNase clive à chaque G), et c) du radical hydroxyle la fragmentation induite par l&#39;ARN est bien au-dessus de fond. La transition de faible à haute Mg (II) est affiliée à diminuer la densité intégrée de la bande individuels et les groupes de bandes indiquant la formation d&#39;ARN structure tertiaire. Simple ou des groupes de nucléotides contigus dont le clivage des changements de façon concomitante sont considérés comme une «protection». Les protections • OH caractéristique du Mg (II) la médiation de pliage de l&#39;ARN P4-P6 correspondent étroitement aux régions inaccessibles solvant de la molécule observée dans sa structure cristalline. 15 Plié molécules d&#39;ARN c<html> une ont des degrés très différents (à savoir, comment proche de fond le déclin bande de densité) de protection contre les contacts tertiaires qui n&#39;ont pas été corrélés avec phénomène structurel ou dynamique claire. Ainsi, certaines molécules d&#39;ARN se montrent bien défini des transitions structurelles, comme le montre la figure 3, et d&#39;autres non. Intensités des bandes sont quantifiés et normalisés par SAFA 12 analyse (figure 3B). La sortie est un &quot;thermique&quot; complot visualiser le degré relatif de protection contre • OH. La transition de couleur décrit les changements d&#39;accessibilité lors de Mg (II) plus. Blanc à rouge ou bleu montre nucléotides plus accessible ou plus protégés, respectivement. Chaque degré d&#39;ombrage est affilié à une valeur numérique qui peut être tracée comme une courbe de protection (figure 3C) et analysées par un modèle de liaison tels que l&#39;équation de Hill (1). La constante d&#39;équilibre de dissociation affilié à la protection de 153 à 155 est à peu près le double de la valeur correspondante de protection 163-164. ONTENU &quot;&gt; <img alt="Figure 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Figure 1. Production de radicaux hydroxyles et P4-P6 pliage ARN. A) le Fe (II) catalyse la production de radicaux hydroxyles par le peroxyde d&#39;hydrogène. L&#39;ascorbate réduit le Fe (III) Retour au fer ferreux. B) En l&#39;absence de Mg (II), aucune structure P4-P6 tertiaire est formé, ce qui permet de pénétrer les radicaux hydroxyles et cliver toutes les positions armature accessible. L&#39;addition de Mg (II) initie pliage de P4 à P6, permettant seulement l&#39;épine dorsale accessible au solvant à être clivée par les radicaux hydroxyles. <img alt="Figure 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Figure 2. Schéma pour une expérience empreinte radical hydroxyle. Un Dephophorylation) et 32 P-5&#39;-end d&#39;étiquetage de l&#39;ARN. B) Purification de 32 P-ARN marqué sur un gel de polyacrylamide dénaturant. C) L&#39;excision de la bande de l&#39;ARN, après l&#39;extraction d&#39;ARN, et précipitation à l&#39;éthanol. D) et Prefoldingrepliement de l&#39;ARN. E) Ajout d&#39;fraîchement préparés mélange réaction de Fenton pour générer les radicaux hydroxyles. F) la séparation fragment d&#39;ARN par électrophorèse sur gel dénaturant de polyacrylamide. G) La quantification de fragments d&#39;ARN par un logiciel de SAFA. <img alt="Figure 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Figure 3. Analyse de fragments d&#39;ARN après réaction empreinte peroxydante Fenton. (A) L&#39;ARN a été exposé à des radicaux hydroxyles et les produits de clivage ont été résolues en utilisant l&#39;électrophorèse sur gel dénaturant de polyacrylamide (PAGE). La photo montre des produits de clivage peroxydante avec une concentration croissante de Mg (II). Voies de référence et de contrôle sont étiquetés. Ce fichier image est l&#39;entrée pour le programme SAFA, qui quantifie le volume de chaque bande. (B) Le «thermique» intrigue générée par la SAFA. (C) Un tableur des valeurs relatives densité de la bande intégrés à la sortie du numéro de nucléotides SAFA est analysée par un modèle de liaison tels que la Colline equatisur (1). Régions de protection ont été choisis en fonction de l&#39;augmentation de la densité de la bande intégrale comme une fonction de Mg (II) de concentration. Le Kd est le Mg (II) la concentration à laquelle la moitié de l&#39;ARN est plié sur le site à surveiller. Le coefficient de Hill, n H, est une mesure de la pente de la courbe qui donne des informations sur la coopérativité de liaison et fournit une estimation plus faible pour le nombre d&#39;ions magnésium impliquée dans le pliage de ce site particulier. <img alt="Tableau 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tableau 1. Volumes Représentant pour générer des échantillons d&#39;ARN contenant différents Mg (II) sur les concentrations.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+SHAPE+and+hydroxyl+radical+analysis+of+RNA+structure+and+ribonucleoprotein+assembly.">High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.</a> Methods. Enzym. 468, 67-89 (2009).</li><li>Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+large+RNA+molecules+with+knowledge-based+potentials+and+structural+filters.">Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters.</a> RNA. 15, 189-199 (2009).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Hydroxyle empreinte radicale est un outil précieux pour évaluer la surface accessible au solvant des acides nucléiques. La formation quantitative et qualitative de la structure tertiaire 14 peut être suivie en fonction de paramètres tels que le type et la concentration d&#39;ions, pH, température, protéines de liaison ou de pliage des co-facteurs. La combinaison convaincante d&#39;un protocole simple et peu coûteux et l&#39;accessibilité résultant de solvant et de l&#39;information pliage sur un seu...

<table border="1">
	<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

Molécules d&#39;ARN jouent un rôle essentiel dans la biologie. En plus de transmettre l&#39;information génétique, l&#39;ARN peut incorporer uniques structures tertiaires remplir un rôle de régulateur biologiques spécifiques, liant ou de catalyseur. Information sur la formation de contacts tertiaire est essentielle pour comprendre la fonction des molécules d&#39;ARN. Les radicaux hydroxyles (OH •) sont des sondes unique de la structure des acides nucléiques en raison de leur grande réactivité et une petite taille. 1 Lorsqu&#39;il est utilisé comme une sonde d&#39;empreinte, les radicaux hydroxyles cartographier la surface accessible au solvant de l&#39;squelette phosphodiester de l&#39;ADN et l&#39;ARN 1 2 avec aussi fine que la résolution seul nucléotide. Hydroxyle empreinte radical peut être utilisé pour identifier les nucléotides au sein d&#39;une surface de contact intermoléculaires, par exemple en ADN-protéines et d&#39;ARN une teneur en protéines complexes. Equilibre et cinétique 3 4 transitions peut être déterminé en effectuant des empreintes hydroxyles radicaux en fonction d&#39;une solutisur la variable ou l&#39;heure, respectivement. Une caractéristique clé de l&#39;empreinte est que l&#39;exposition limitée à la sonde (par exemple, &quot;la cible unique cinétiques») des résultats de l&#39;échantillonnage uniforme de chaque nucléotide du polymère. 5 Dans cet article, vidéo, nous utilisons le domaine P4-P6 du ribozyme de Tetrahymena pour illustrer la préparation des échantillons d&#39;ARN et la détermination d&#39;un Mg (II)-médiée isothermes pliantes. Nous décrivons l&#39;utilisation du protocole bien connu l&#39;empreinte de radicaux hydroxyles qui nécessite H 2 O 2 (nous appelons cela la &quot;peroxydante« protocole) et une valeur, mais pas très connu, alternative qui utilise naturellement dissous O 2 (nous appelons cela «l&#39; oxydatif &quot;protocole). Un aperçu de la réduction des données, la transformation et les procédures de l&#39;analyse est présentée.

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 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Ce protocole décrit comment quantifier les Mg (II)-dépendante de la formation de l&#39;ARN structure tertiaire par deux méthodes d&#39;empreinte radical hydroxyle.

Les changements d&#39;équilibre dans la structure de l&#39;ARN de surveillance par «peroxydation» et «oxydatif» Hydroxyl Footprinting Radical

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

14.2K vues.  Hunter College. Ce protocole décrit comment quantifier les Mg (II)-dépendante de la formation de l'ARN structure tertiaire par deux méthodes d'empreinte radical hydroxyle.

Vidéo: 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.

Méthodes Single Molecule pour surveiller les changements dans les propriétés élastique bicouche

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

Recherche

Biologie

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.

Surveillance des changements dynamiques dans les niveaux de calcium mitochondrial cours de l&#39;apoptose en utilisant un capteur de calcium codées génétiquement

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.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

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.

À long terme des essais de toxicité létale avec le Crustacé 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.

Isolement de la chromatine par purification d&#39;ARN (CHIRP)

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

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.

ARN prédiction de la structure secondaire via SHAPE à haut débit

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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.

Reconstitution fonctionnelle et Activité du Chan mesures de type sauvage purifiée et Mutant protéine CFTR

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.

Suivi des modifications induites par la drogue dans des récepteurs post-internalisation traite par Analyse 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.

Fecal glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

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

Les changements d'équilibre dans la structure de l'ARN de surveillance par «peroxydation» et «oxydatif» Hydroxyl Footprinting Radical

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