Diese Arbeit wurde durch Zuschüsse aus dem National Institute of Health RO1-GM085130 und National Science Foundation MCB0929394 unterstützt. Wir danken Dr. Marion Schmidt für ihre Gastfreundschaft und für die Erlaubnis zu filmen in ihrem Labor.

1. Vorbereitung der Reagenzien Footprinting <ol><li> Bereiten Sie einen 10x Reaktionspuffer mit 100 mM Natriumcacodylat, 1 mM EDTA und 1 M KCl. Den pH-Wert auf 7,4. Filtern Sie die Puffer unter Verwendung eines 0,2 uM Acetat-Filter-Gerät (Nalgene). Bemerkung: nicht pipet RNA direkt in 10x Puffer. </li><li> Bereiten Sie die Titrationsreaktion Mix für jede Reaktion, wie in Tabelle 1 angegeben. Das Volumen der Titration Mix (1x Puffer und Mg (II) in der gewünschten Konzentration) sollte 90 ul werden, bevor man 10 &amp;mgr; l des RNA in 1x Puffer. </li><li> Bereiten Sie eine RNAse T1 Verdauung Puffer 6.63M Urea, 20 mM Natriumcitrat, 1mM EDTA, 0,25 ug / ul tRNA, 0,025% Xylencyanol und 0,025% Bromphenolblau. Dieser Puffer kann bei 4 ° C gelagert werden bis zu 6 Monaten. </li><li> Die Fenton-Reaktion erfordert frisch zubereiteten wässrigen Lösungen von 250 mM Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA und 500 mM Natrium-L-Ascorbat. Die Konzentrationen festgestellt in Kapitel 10,5 sind optimal für equilibrium-Experimente in Puffer wie Natriumcacodylat, die nicht abzufangen ist Radikalen und diverse zweiwertigen Ionen-Konzentrationen. </li><li> Reagenzien für die peroxidative (1.5.1) und oxidative (1.5.2) footprinting Reaktion. <ol><li> Die peroxidative Fenton Reaktion erfordert frisch zubereiteten wässrigen Lösungen von 100 mM Fe-EDTA-Lösung und 50 mM Natrium-L-Ascorbat und 1,5% H 2 O 2. Die Fe-EDTA-Lösung wird durch Mischen von Fe (NH 4) 2 (SO 4) 2 mit EDTA bereit, das 1,1-fache über EDTA über Eisen haben. </li><li> Kurz vor Eröffnung des oxidativen Spaltung (3.4.2), erstellen Sie die Fenton-Reaktion-Mix durch die Kombination von 2 ul Fe (NH 4) 2 (SO 4) 2 (250 mM), 2,2 ul EDTA (250 mM), 62,5 ul Natrium-L-Ascorbat (500 mM), 22,5 ul 10x Reaktionspuffer und 135,5 ul H 2 O auf die Endkonzentrationen im footprinting Reaktionsgemisch (3.4.2) von 0 zu erreichen.10 mM, 0,11 mM, und 6,61 mM. </li></ol></li></ol> 2. RNA-Präparation für Footprinting Experiment <ol><li> RNA kann durch Standard-in vitro-Transkription der DNA-Vorlagen 6 hergestellt werden oder, wenn kürzer als 50 Nukleotide, erworben (z. B. bei Integrated DNA Technologies, www.idtdna.com) werden. Die empfohlenen Labor Sauberkeit Verfahren zur Probe Integrität der RNA während Footprinting Experimente zu erhalten. 7 </li><li> Dephosphorylierung 8 von in vitro transkribierte RNA ist notwendig, vor 32 P Kennzeichnung der 5 &#39;Ende der RNA. 10 pmol RNA werden durch kinasing endmarkiert. 9 Die Lösung sollte nach erfolgter Inaktivierung der Phosphatase bewölkt. </li><li> Purify radioaktiv markierte RNA mit denaturierende Gelelektrophorese. Die RNA wird durch zwei anschließende Gel Extraktion mit 0,3 M Natriumacetat (pH 5,5) erholt. RNA wird durch Mischen von 0,5 ml wässrige Lösung mit 1 ml Ethanol und anschließender ausgefälltInkubation für 1 Minute auf Trockeneis. Spin Probe bei 4 ° C, 12500 rpm für 0,5 Stunden. Überstand verwerfen und waschen RNA-Pellet mit 70% Ethanol. Entfernen Sie nach dem zusätzlichen Zentrifugationsüberstand und trocken Pellet im Vakuum. </li><li> Lösen Sie die 32 P-markierten RNA-Proben in 330 ul 1x Buffer.Pipet 30 ul der RNA-Lösung in einem Reaktionsrohr, Niederschlag mit Ethanol und trockene RNA im Vakuum; halten dieses Rohr für die Erzeugung der Referenz Leiter (Schritt 3.5). Denaturieren gepuffert RNA-Lösung durch Erhitzen bei 95 ° C für 2 min. Kühlen Sie die Proben auf Raumtemperatur für 15 min und geben einen schnellen Spin zur Senkung der Kondensate auf den Deckeln der Rohre wieder in Lösung. </li></ol> 3. Die Footprinting Experiment <ol><li> Die Elektrophorese Kamm bestimmt die maximale Anzahl der Datenpunkte pro analytischen Gel. Wir verwenden eine 30 gut kämmen. Die Referenz Leiter belegt 2 Brunnen und die ungespaltenen Kontrolle ein. Bereiten 27 Reaktionsgefäße. Die endgültige Mg (II) concentrations sollten gleichmäßig auf einer logarithmischen Skala Spanning Größenordnungen um die Titration Mittelpunkt angeordnet werden. </li><li> Separat inkubieren RNA und Mg (II)-Lösungen bei 50 ° C für 5 min. Mix 10 ul RNA-Lösung mit 90 ul der entsprechenden Menge an Mg (II) in 1x Puffer auf ein Endvolumen von 100 ul zu erreichen. Inkubieren Sie die Lösungen für 30 min bei 50 ° C. </li><li> Lassen Sie die Lösungen bei 25 ° C für 1 Stunde ausgleichen, während Faltung auftritt. </li><li> Peroxidativer (3.4.1) oder oxidative (3.4.2) Hydroxylradikal footprinting Reaktion. <ol><li> Starten Sie den peroxidative Reaktion, indem Tröpfchen von 2 ul Fe-EDTA (100 mM), 2 ul Natrium-L-Ascorbat (50 mM) und 2 ul H 2 O 2 (1,5%) von einander an der Spitze in der Reaktion abgetrennt Röhrchen mit der RNA-Lösung und starten Sie die footprinting Reaktion durch kräftiges Mischen. Stoppen der Reaktion nach 15 Sekunden durch Zugabe von 300 ul kaltem absolutem Ethanol. Drehen Sie den Rohren 3-5 mal. Niederschlag, Wasch-undtrockene Pellet, wie in Schritt 2.4. </li><li> Starten Sie die oxidative Hydroxylradikal footprintingreaction durch Zugabe von 5 ul der frisch zubereiteten Fenton-Reaktion-Mix (1,6). Inkubieren für 30 min bei 25 ° C. Add 300 pl kaltem absolutem Ethanol, um die Reaktion zugeben und durch Drehen der Rohre 3-5 mal stillen. Niederschlag, waschen und trocknen das Pellet wie in Schritt 2.4. </li></ol></li><li> Generieren Sie eine RNase T1 verdauen Referenzprobe nach üblichen Verfahren 10 mit der Lösung in Schritt 1.3 vorbereitet. </li><li> Lösen RNA-Pellets in 8 ul Gel Loading Dye II (Ambion) und bestätigen RNA wird durch die Verwendung der Geiger-Zähler resuspendiert. </li><li> Bereiten Sie eine Denaturierung, 8% Polyacrylamid Sequenzierungsgel nach Standardprotokollen. 11 Legen Proben, darunter zwei Referenzen und eine Kontrollgruppe. Separate RNA-Fragmente bei 60 W - 75 W für 2,5 Stunden. Expose getrockneten Gel zu einer Speicherung Leuchtschirm über Nacht. Scan Leuchtschirm mit einem Imaging-System für filmlosen Autoradiographie, z. B.. Sturm 865 (GE Healthcare) oder Taifun (GE Healthcare). Transfer-Gel-Image-Datei auf Ihren Computer. </li></ol> 4. Data Analysis <ol><li> Das Hydroxyl-Radikal Reaktivität jedes Nukleotid ist durch Quantifizierung der Intensität jeder Band auf der analytischen Gel bestimmt. Downloaden, installieren und öffnen SAFA, ein einfach zu Open-Source-Software für Single-Band-Montage und Quantifizierung verwenden. 12, 13 Befolgen Sie die Bedienungsanleitung beachten. Kurz gesagt, laden Sie die RNA-Sequenz als. Txt-Datei, gefolgt von der Gel-Bild als. Gel-Datei. Passen Bandenintensitäten, definieren die Gassen, wählen Sie einen Anker Spur, und führen Sie ein Gel Ausrichtung. Zuordnung der Banden zu Nukleotiden tritt in Bezug auf RNase T1 Verdauung Leiter. Quantifizierung der Band Integrale und die Normalisierung / colorplot Funktion zu normalisieren und weisen potenziell invariant Rückstände. Speichern Sie die Ausgabe als. Txt-Datei. </li><li> SAFA gibt eine Tabelle mit Spalten repräsentieren Spuren auf dem Gel und Zeilen, die die inintegrierte Band Dichte der einzelnen Banden, die die RNA-Fragment. Zunächst identifizieren die Schutzgebiete, die eine merkliche Veränderung in Lösungsmittelzugänglichkeit Display durch einen Vergleich der Schutz-Profil aus der kein Mg (II)-Probe mit dem Profil des Endpunktes Mg (II)-Konzentration (50 mM in unserem Beispiel) abgeleitet. Je niedriger der Wert, desto mehr schützten die Nukleotid ist gegen den Angriff von Hydroxyl-Radikalen und umgekehrt. </li><li> Create Falten Isothermen aus den normalisierten Band Integrale von einzelnen oder Gruppen von Nukleotiden im Vergleich zu Mg (II)-Konzentration. Die Isothermen sind individuell auf fraktionelle Sättigung durch fi = L + (U - L) skaliert • <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> wobei f die integrierte Dichte der Band (s) bezeichnet, die analysiert werden, stellen L-und U die untere und obere Grenzen für die Übergangs-und <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> ist fraktionelle Sättigung. 14 Die Daten sind fit mit einem nicht-linearen lOsten Quadrat-Analyse-Programm (Wir verwenden entweder Origin (OriginLab) oder GraphPad (GraphPad Software, Inc.).), um die Hill-Gleichung (1), <img alt="Gleichung 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> wo K d ist das Gleichgewicht Dissoziationskonstante, [M] entspricht der Konzentration der Variablen, die die Faltung Reaktion vermittelt, Mg (II) in diesem Beispiel, und n H ist der Hill-Koeffizient. Dieses Verfahren skaliert die Umstellung auf fraktionelle Sättigung bestimmt seinen Mittelpunkt und bietet eine phänomenologische Test, ob ein Übergang sigmoidal. Isothermen von Sequenziergelen (Abbildung 3A), die von SAFA (Abbildung 3B) wurden analysiert und fit wie oben beschrieben abgeleitet sind in Abbildung 3C gezeigt. Die fraktionelle Sättigung Isothermen in Abbildung 3 dargestellt wurden durch Skalierung in einer Tabellenkalkulation erstellt die Werte von SAFA gegen die besten passen Ober-und Untergrenzen (U-und L) mit der Gleichung Bruchteil generiert = value / (U - L) - L / (U - L). </li></ol> 5. Repräsentative Ergebnisse: Abbildung 3 zeigt repräsentative Ergebnisse aus P4-P6-RNA • OH Footprinting Experimente. Das Gel-Bild (links) zeigt an, dass a) Hintergrund Spaltung minimal ist (rechte Spur), b) single nucleotide Zuordnung ist möglich durch gut definierte Banden in den T1 Spur (RNase T1 spaltet bei jedem G), und c) das Hydroxyl-Radikal induzierten Fragmentierung der RNA ist weit über Hintergrund. Der Übergang von niedrigen zu hohen Mg (II) wird mit abnehmender integrierte Band Dichte der einzelnen und Gruppen von Bändern auf die Bildung von RNA Tertiärstruktur angeschlossen. Einzel-oder Gruppen von aufeinander folgenden Nukleotid deren Spaltung Änderungen mitverwendet werden als &quot;Schutz&quot; bezeichnet. Die • OH-Schutz Merkmal der Mg (II)-vermittelte Faltung der P4-P6-RNA in engem Zusammenhang mit dem Lösungsmittel unzugänglichen Regionen des Moleküls in seine Kristallstruktur beobachtet entsprechen. 15 Gefaltete RNA-Moleküle c<html> ein sehr unterschiedlichem Ausmaß (dh, wie Hintergrund schließen die Band Dichten Rückgang) des tertiären Berührungsschutz, die nicht mit klaren Struktur-oder dynamisches Phänomen wurden korreliert. So werden einige RNA-Moleküle zeigen, gut definierte strukturelle Übergänge, wie in Abb. 3 gezeigt, und manche nicht. Band Intensitäten quantifiziert und durch SAFA 12 Analyse (Abbildung 3B) normiert. Die Ausgabe ist eine &quot;thermische&quot; Plot Visualisierung der relativen Schutz gegen • OH. Der Farbübergang beschreibt die Zugänglichkeit zu ändern, auf Mg (II) hinaus. Weiß auf Rot oder Blau zeigt mehr zugänglich oder mehrere geschützte Nukleotide, jeweils. Jedes Grad der Schattierung wird mit einem numerischen Wert, der als Schutz-Kurve (Abbildung 3C) aufgetragen werden können und von einem Binding-Modell wie der Hill-Gleichung (1) angeschlossen. Die Gleichgewichts-Dissoziationskonstante mit Schutz 153-155 angegliedert ist etwa doppelt so hoch wie der entsprechende Wert des Schutzes 163-164. NHALT &quot;&gt; <img alt="Abbildung 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Abbildung 1. Hydroxylradikal Produktions-und P4-P6 RNA-Faltung. A) Fe (II) katalysiert die Bildung von Hydroxylradikalen aus Wasserstoffperoxid. Ascorbat reduziert Fe (III) zurück zu Eisen-II. B) In Abwesenheit von Mg (II), ist kein P4-P6 Tertiärstruktur gebildet, so dass Hydroxyl-Radikale zu durchdringen und zu spalten alle zugänglich Backbone-Positionen. Die Zugabe der Mg (II) initiiert Faltung des P4-P6, so dass nur das Lösungsmittel zugänglich Rückgrat durch die Hydroxyl-Radikale gespalten werden. <img alt="Abbildung 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Abbildung 2. Für einen Hydroxyl-Radikal footprinting Experiment Outline. A) Dephophorylation und 32 P-5&#39;-Ende Markierung von RNA. B) Reinigung von 32 RNA auf einem denaturierenden Polyacrylamidgel P-markierte. C) Exzision von RNA-Band, anschließende RNA-Extraktion und Ethanolfällung. D) Vorfaltung undFaltung der RNA. E) Die Zugabe von frisch zubereiteten Fenton Reaktionsgemisch zur Erzeugung der Hydroxyl-Radikale. F) RNA-Fragment Trennung durch denaturierende Polyacrylamid-Gelelektrophorese. G) Quantifizierung von RNA-Fragmenten durch SAFA-Software. <img alt="Abbildung 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Abbildung 3. Analyse von RNA-Fragmenten nach peroxidative Fenton footprinting Reaktion. (A) Die RNA wurde zu Hydroxyl-Radikalen ausgesetzt und die Spaltprodukte wurden gelöst mit denaturierenden Polyacrylamid-Gelelektrophorese (PAGE). Das Bild zeigt peroxidative Spaltprodukte mit zunehmender Konzentration von Mg (II). Reference und Kontrolle Gassen beschriftet sind. Diese Image-Datei ist der Eingang für das SAFA-Programm, das die Lautstärke der einzelnen Bänder quantifiziert. (B) Die &quot;thermische&quot; Verschwörung von SAFA generiert. (C) eine Tabelle mit Werten über integrierte Band Dichte an Nukleotid-Nummer Ausgabe von SAFA wird durch ein verbindliches Modell wie der Hill equati analysiertauf (1). Regionen des Schutzes wurden nach der Erhöhung der Band integralen Dichte als Funktion der Mg (II)-Konzentration gewählt. Die K d ist die Mg (II)-Konzentration bei der die Hälfte der RNA auf der Baustelle überwacht gefaltet ist. Der Hill-Koeffizient, n H, ist ein Maß für die Steigung der Kurve, die Auskunft über die Bindung Kooperativität und bietet eine niedrigere Schätzung für die Zahl der Magnesium-Ionen in Falten von diesem besonderen Ort beteiligt. <img alt="Tabelle 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tabelle 1. Repräsentativen Mengen zur Erzeugung von RNA Proben mit verschiedenen Mg (II)-Konzentrationen.

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Keine Interessenskonflikte erklärt.

Hydroxyl-Radikal Footprinting ist ein wertvolles Instrument, um das Lösungsmittel zugängliche Oberfläche von Nukleinsäuren zu beurteilen. Qualitative und quantitative Bildung der Tertiärstruktur 14 kann als eine Funktion von Parametern wie Ionen-Art und Konzentration, pH, Temperatur, bindende Proteine ​​oder Falten Co-Faktoren zu beachten. Die überzeugende Kombination aus einem geradlinig und kostengünstige Protokoll und die daraus resultierende Lösungsmittel Zugänglichkeit und Falten Information...

<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

RNA-Moleküle spielen eine wesentliche Rolle in der Biologie. Neben der Übertragung genetischer Information, kann RNA in einzigartige Tertiärstrukturen Erfüllung einer bestimmten biologischen Rolle als Regulierer, Bindemittel oder Katalysator zu falten. Informationen über tertiäre Bildung Kontakt ist unerlässlich, um die Funktion von RNA-Molekülen zu verstehen. Hydroxyl-Radikale (• OH) sind einzigartig Sonden der Struktur von Nukleinsäuren aufgrund ihrer hohen Reaktivität und geringen Größe. 1 Wenn ein footprinting Sonde verwendet, map Hydroxylradikale das Lösungsmittel zugängliche Oberfläche des Phosphodiester-Rückgrat der DNA 1 und RNA 2 mit so fein wie single nucleotide Auflösung. Hydroxyl-Radikal footprinting können die Nukleotide innerhalb einer intermolekularen Kontaktfläche, in DNA-Protein-1 und RNA-Protein-Komplexe z. B. zu identifizieren. Equilibrium 3 und 4 kinetische Übergänge können durch die Durchführung Hydroxylradikal Footprinting als Funktion einer Lö bestimmt werdenauf variable oder Zeit, bzw.. Ein wesentliches Merkmal des Fußabdrucks ist, dass eine begrenzte Exposition mit der Sonde (zB &quot;Single-Hit-Kinetik&quot;) führt in die einheitliche Probenahme jedes Nukleotid des Polymers. 5 In diesem Video-Artikel verwenden wir die P4-P6-Domäne des Tetrahymena Ribozym RNA Probenvorbereitung und die Bestimmung eines Mg (II)-vermittelte Faltung Isothermen zeigen. Wir beschreiben die Verwendung der bekannten Hydroxyl-Radikal footprinting Protokoll, das H 2 O 2 (wir nennen dies den &quot;peroxidative&quot;-Protokoll) und eine wertvolle, aber nicht allgemein bekannt, dass alternative natürlich gelösten O 2 verwendet (wir nennen so bedarf dies der &#39; oxidative &#39;Protokoll). Eine Übersicht über die Datenreduktion, Transformation und Analyse-Verfahren vorgestellt.

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

Dieses Protokoll beschreibt, wie die Mg (II)-abhängige Bildung von RNA Tertiärstruktur durch zwei Methoden der Hydroxyl-Radikal footprinting quantifizieren.

Überwachung Equilibrium Veränderungen im RNA-Struktur von &quot;peroxidativer&quot; und &quot;Oxidativer &#39;Hydroxylradikal Footprinting

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 Aufrufe.  Hunter College. Dieses Protokoll beschreibt, wie die Mg (II)-abhängige Bildung von RNA Tertiärstruktur durch zwei Methoden der Hydroxyl-Radikal footprinting quantifizieren.

Serie: 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.

Single Molecule Methoden zur Überwachung von Veränderungen 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 ...

Forschung

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.

Überwachung dynamischen Veränderungen in der mitochondrialen Kalzium während der Apoptose mit einem genetisch kodierten Kalzium-Sensor

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Langfristige letale Toxizität Test mit dem Krustentier 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.

Chromatin Isolierung von RNA-Reinigung (Chirp)

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.

Nachweis viraler RNA durch Fluoreszenz<em&gt; In-situ-</em&gt; Hybridisierung (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 Secondary Structure Prediction Mit 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 ...

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.

Funktionelle Rekonstitution und Kanalaktivitätsmessungen von gereinigtem Wildtyp und mutierten CFTR-Protein

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.

Tracking-Drug-induzierte Veränderungen der Post-Rezeptor-Internalisierung Handel durch Colocalizational Analysis

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 Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

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