This work was supported, in whole or in part, by NIH Grants R01 AA 020758-04, U01DK 061731-13 and T32 DK 007150-38 to AJS and T32 DK 007150-38 to AA. This is original work and is not under consideration elsewhere for publication.

1. Entnahme von Blutproben <ol><li> Sammeln peripheren Fasten Blutproben in einem 10 ml-Plastikröhrchen mit Antikoagulans (EDTA) durch Standard venipuncture einer prominenten Vene in der Ellenbeuge (die mehrere Vorteile gegenüber anderen Antikoagulantien hat). </li><li> Zentrifugieren der Blutproben bei 1.600 xg für 20 min bei 4 ° C in einer Tischzentrifuge Plasma frei von roten Blutkörperchen und geringe Mengen an RNA zu erhalten. </li><li> zentrifugieren sequentielles der Überstand bei 3.000 g (4 ° C) in einem Schwingbecher-Rotor für 10 min zu entfernen WBC &amp; Platelets und dann weitere 15 min jeweils verbleibenden Zelltrümmer zu entfernen. </li><li> Messen der Dichte des Plasmas mit einem Densitometer bei RT unter Verwendung gemäss Herstellungsanweisungen. HINWEIS: Die Einstellung der Dichte (d = 1,023 g / ml) mit 0,9% Kochsalzlösung kann nach der Entfernung von Exosomen erforderlich sein, jedoch vor der Dichtegradient Ultrazentrifugation. </li></ol&gt; 2. exosome Entfernen von Plasma <ol><li> Entfernen Sie die zirkulierenden Exosomen , die eine ähnliche Dichte wie HDL und eine quantitativ signifikante Quelle von miRNA 3 darstellen. <ol><li> Dies geschieht durch Zugabe von 252 &amp; mgr; l exosome Fällungslösung zu 1 ml Plasma, gefolgt von Inkubation für 30 min bei 4 ° C. Um die Exosomen Pellet aus Zentrifugation der Mischung für 30 min bei 1500 g bei 4 ° C. </li><li> Zur Isolierung HDL, je 1 ml des resultierenden Überstand auf eine Polycarbonat-dickwandigen Ultrazentrifugenröhrchen für die weitere Verarbeitung mit Dichte-Ultrazentrifugation (siehe unten). </li></ol></li></ol> 3. Dichte-Ultrazentrifugation (Abbildung 1). <ol><li> Separater HDL verwenden, um ein 3-Stufen-Prozess einen Boden mit einem Ultrafestwinkelrotor, der bei 448.811 x G und 8 ° C, jeweils eingesetzt wird. </li><li> Bereiten Sie drei unterschiedliche Dichte Lösungen nacheinander und frisch für jede Isolation. <ol><li> Bereiten Lösung A (Isolation von VLDL, d = 1,006 g / ml) durch Auflösen von 11,4 g NaCl (NaCl: 0,195 mol), 0,1 g EDTA2Na und 1 ml 1N NaOH in 1000 ml autoklaviertem destilliertem Wasser. Dann fügen Sie eine zusätzliche 3 ml autoklaviert-destilliertes Wasser. </li><li> Vorbereitung der Lösung B (Isolierung von LDL, d = 1,182 g / ml) durch Zugabe von 25,2 g NaBr 100 ml Lösung A (NaCl 0,195 mol, NaBr 2,44 mol). </li><li> Bereiten Lösung C (Isolierung von HDL, d = 1,470 g / ml) durch Mischen von 78,8 g NaBr mit 100 ml Lösung A (NaCl 0,195 mol, NaBr 7,7 mol). Bestätigen Sie die entsprechende Dichte bei RT unter Verwendung eines Densitometers. Halten Sie alle Lösungen bei 4 ° C bis zur weiteren Verwendung. </li></ol></li></ol> 4. Isolierung von VLDL <ol><li> Mischen Sie 1 ml Plasma (durchschnittliche Dichte = 1,023 g / ml) und nukleasefreiem 200 ul Fat Red 7B in einem 6,5 ml Polycarbonat dickwandigen Ultrazentrifugenröhrchen. </li><li> Dann Schicht vorsichtig 5 ml der Lösung A auf die Oberseite des Gemisches. Falls erforderlich, fügen Sie zusätzliche Fat Red 7B auf der Lösung A to das Gewicht jedes Rohr balancieren. Zentrifuge für 2 h (Beschleunigung - 5), (Verzögerung - 7). HINWEIS: Während der Zentrifugation werden die Lipoproteine ​​als Band auf ihre Gleichgewichtsdichte Regionen akkumuliert. </li><li> Am Ende der Lauf beobachten 2 Schichten. Entfernen Sie 1,5 ml der VLDL-Fraktion, die die obere Schicht und lagern bei 4 ° C. </li><li> Schließlich mit einer Pipette Übertragungs 4 ml vom Boden des Röhrchens das LDL enthält, HDL, Albumin und Fettsäure-Fraktion zu einem neuen Polycarbonatrohr für LDL Isolation. </li></ol> 5. Isolierung von LDL <ol><li> Mix 2 ml Lösung B und 100 ul nukleasefreiem Fat Red 7B in das Röhrchen mit dem LDL und HDL-Fraktion (Abschnitt 4) sind. </li><li> Zentrifuge Dann aus 3 h (Beschleunigung 9, Verzögerung 7). Danach entfernen 1,5 ml der LDL-Fraktion die oberste Schicht darstellt und halten bei 4 ° C oder bei -80 ° C. Schließlich übertragen 4 ml vom Boden des Röhrchens das HDL enthaltendeFraktion auf eine neue Polycarbonatrohr. </li></ol> 6. Isolierung von HDL <ol><li> Mix 2 ml Lösung C, 100 &amp; mgr; l Nuklease frei Fat Red 7B und 15 ul 98% β-Mercaptoethanol in das Rohr die HDL-Fraktion enthält, respectively. </li><li> Zentrifuge für 3 h (Beschleunigung 9, Verzögerung 7). Danach 2 ml der HDL-Fraktion zu entfernen die oberste Schicht darstellt und entweder halten bei 4 ° C oder bei -80 ° C. </li></ol> 7. Die Entsalzung und Konzentration von Lipoprotein-Fraktionen <ol><li> Um Interferenzen mit anschließender Agarose-Gelelektrophorese und PCR zu vermeiden, entfernen übermäßige Salz während Dichtegradient Ultrazentrifugation hinzugefügt unter Verwendung von Zentrifugal-Filtervorrichtungen mit dem geeigneten Molekulargewichtsgrenze (3K Rohr für VLDL und 10K Rohr für LDL / HDL), wie durch die Anweisungen des Herstellers beschrieben ist. <ol><li> Kurz gesagt, nach 2,5 ml kaltem PBS (137 mM NaCl, 2,7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7,4) Zugabe centrifuge der gesamte VLDL-Fraktion gesammelt während Dichtegradient Ultrazentrifugation bei 4 ° C für 60 min eine Schwingbecherrotor verwendet wird. </li><li> Desalt die LDL-Fraktion zweimal mit 10 ml eiskaltem PBS für jeweils 30 min. Verwenden Weiter wurden 13 ml eiskaltem PBS zweimal für Entsalzen der HDL-Fraktion. Je höher PBS Volumen ist notwendig, die Mobilität mit Agarose-Gelelektrophorese zu verbessern. Nach der Zentrifugation entfernen bei 4 ° C oder bei -80 ° C gelagert, die Lipoprotein enthält, gelöste Stoffe und zu halten. </li></ol></li></ol> 8. Agarose-Gelelektrophorese <ol><li> Perfrom Lipoprotein Agarosegelelektrophorese das Kit mit kleineren Modifikationen von den Anweisungen des Herstellers verwendet wird, wie folgt. HINWEIS: Dieser Schritt ist nur die Qualität und Reinheit der konzentrierten Lipoprotein Proben zu bewerten ist. <ol><li> Kurz gesagt, erhalten 6 ul des entsalzten Lipoprotein-Fraktion mit Ultrazentrifugation Dichte und laden auf einen vorgegossenen Lipoprotein-Gel. Verwenden menschlichen lipoprotein Standards für VLDL, LDL und HDL als Größenreferenz. Führen Sie die Elektrophorese bei RT bei 100 V für 60 min unter Verwendung von Rep Prep-Puffer. </li><li> Trocknen des Gels für 10 min und dann Färbung für 10 min bei RT mit Fat Red 7B. Entfärben des Gels in einem Gemisch aus Methanol-Wasser 75:25 (v / v) und trockenem erneut 5 min. </li></ol></li></ol> 9. RNA Extraktion und Reinigung <ol><li> Perfrom Isolierung von miRNA durch gereinigtes humanes HDL mit dem Serum / Plasma-miRNA Isolierung und Reinigung Kit. <ol><li> Kurz gesagt, 1 ml RNA Lysereagenz bis 200 &amp; mgr; l gereinigtem HDL, mit einem Vortexer mischen und dann für 5 min bei RT inkubiert, um die vollständige Dissoziation der Nucleoprotein-Komplexe und Inaktivierung von RNasen gewährleisten. </li><li> Dann Spike 3,5 ul synthetischer Caenorhabditis elegans microRNA (cel-miR-39, 1,6 x 10 8 Kopien / ul) in die Mischung. Führen Sie dann nach RNA-Extraktion aus den Anweisungen des Herstellers. </li></ol></li><li> Führen Sie purificatiauf der extrahierten-miRNA mit elute Spin-Säulen gemäß den Anweisungen des Herstellers. Messen Sie die Konzentration von miRNA aus gereinigtem HDL mit einem spectrophorometer. HINWEIS: Die Elution von miRNA aus den Spin-Säulen verwendet 16 &amp; mgr; l RNase-freiem Wasser. </li></ol> 10. Reverse Transkription (RT-PCR) <ol><li> Isolieren 100 ng des miRNA aus HDL gespikt mit synthetischen miRNA (cel-miR-39) und revers transkribiert in einem 20 ul Reaktionsvolumen der reversen Transkription unter Verwendung von Kits und gemäß den Anweisungen des Herstellers. </li><li> Führen Sie entsprechende Kontrollen ohne Vorlage miRNA (NTC) und ohne reverse Transkriptase-Enzym-Mix (NRT). </li></ol> 11. Real-time PCR (qRT-PCR) <ol><li> Perfrom PCR in Echtzeit in einem Gesamtvolumen von 20 &amp; mgr; l mit 2 &amp; mgr; l einer 1: 2 Verdünnung der cDNA, 10 &amp; mgr; l PCR-Mix, 2 &amp; mgr; l Universal-Primer, 2 &amp; mgr; l miRNA Primern und 4 &amp; mgr; l RNase-freiem Wasser. </li><li> Führen Sie die Reaction in 96-Well-Platten bei 95 ° C für 15 min, von 45 Zyklen von 94 ° C für 15 sec und 55 ° C für 30 s und eine Verlängerungsphase bei 70 ° C für 30 s.ec alle Reaktionen in Triplikaten perfrom . </li><li> Als nächstes calcuate relativen Mengen von miRNA durch die 2 -ΔΔ Ct Methode nach der Normalisierung auf das synthetische Housekeeping - Gen unter Verwendung gemäß den Anweisungen des Herstellers. </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+are+transported+in+plasma+and+delivered+to+recipient+cells+by+high-density+lipoproteins.">MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.</a> Nat Cell Biol. 13 (4), 423-433 (2011).</li><li>Wagner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+levels+and+cellular+transfer+of+circulating+lipoprotein-bound+microRNAs.">Characterization of levels and cellular transfer of circulating lipoprotein-bound microRNAs.</a> Arterioscler Thromb Vasc Biol. 33, 1392-1400 (2013).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+185,+96,+and+223+repress+selective+high-density+lipoprotein+cholesterol+uptake+through+posttranscriptional+inhibition.">MicroRNAs 185, 96, and 223 repress selective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition.</a> Mol Cell Biol. 33 (10), 1956-1964 (2013).</li><li>Rayner, K. 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Die Autoren haben nichts zu offenbaren.

Identifizierung neuer Biomarker aus Blut werden in der klinischen Diagnose und Prognose von verschiedenen Krankheiten unterstützen. MicroRNAs sind bekannt , alle Qualitäten von Biomarkern zu besitzen und haben in verschiedenen Studien 14-17 gezeigt. In dieser Studie haben wir eine schnelle und einfache einfache Methode zur Isolierung miRNA aus Plasma-HDL demonstriert. Herkömmliche Dichtegradient Ultrazentrifugation Verfahren zur Isolierung von VLDL, LDL und HDL , hängt von genauen Abtastung von Plasma, pr...

MicroRNAs are endogenous non-coding tiny RNA species that are highly conserved and are considered key players in the regulation of various biological processes by degrading or repressing specific target messenger RNAs1. Because miRNAs act intracellularly they have been explored as tissue-derived biomarkers which led to the discovery of tissue-specific functions of these miRNA. However, miRNAs are also found extracellularly either associated with proteins or in exosomes/micro vesicles that effectively can shield them from degradation by extracellular RNases2. More recent studies have shown that the protective effect of HDL may not be closely linked to its capability to promote cholesterol efflux but rather to its non-cholesterol cargo, in particularly as a circulating miRNAs carrier 3, 4. These miRNAs may not only modulate lipid metabolism but are also associated with anti-inflammatory, antioxidant and antithrombotic effects of the HDL-miRNA complex 5, 6.
To further explore the role of miRNAs carried in HDL particles, a simple and easy protocol needs to be established for miRNA extraction from isolated highly purified HDL for use in clinical routine. Numerous methods have been described to isolate HDL. These methods are either very time consuming or require large volume of plasma that may require sample pooling, extensive dialysis for desalting isolated lipoproteins and they do not completely remove exosomes as a source of miRNAs3, respectively. Here we describe a simple and rapid method that can isolate miRNA from highly purified HDL utilizing small volume of blood samples on a larger scale. We believe that this method may serve as good reference to promote research into the role of circulating miRNAs and in particular the role of HDL in facilitating communication between various cells and organs.

<table border="0" cellpadding="0" cellspacing="0" width="965">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">Plastic Vacutainer Lavender K2EDTA tubes&#160;</td>
			<td style="width:279px;">Becton, Dickinson and Company</td>
			<td style="width:172px;">366643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific, Sorvall Legend X1R&#160;</td>
			<td style="width:172px;">75004261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Densito 30PX densitometer</td>
			<td>Mettler Toledo</td>
			<td style="width:172px;">MT51324450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoQuick solution&#160;</td>
			<td>Invitrogen</td>
			<td style="width:172px;">4484451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate thick-walled ultracentrifuge tube</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">O3237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall WX100 ultracentrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">46902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fat Red 7B&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;">201618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal filter devices 10K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-centrifugal filter devices 3K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC800308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickGel Lipo kit&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3344,3544T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human lipoprotein standards for VLDL, LDL and HDL</td>
			<td>LipoTrol; Helena Laboratories</td>
			<td style="width:172px;">5069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rep Prep buffer&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MinElute spin columns&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 1000 analyzer</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miScript II RT Kit&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;">218161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX96 Touch real-time PCR detection system</td>
			<td>BioRad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Kit</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">217184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript Primer Assays</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">141078139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript SYBR Green PCR Kit&#160;</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">218073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Spike-In Control</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">219610</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

Isolierung von High Density Lipoprotein Nach Entfernen von Exosomen MiRNA aus hochgereinigtem HDL zu erhalten , ist es notwendig , Exosomen zu entfernen , die eine Quelle der Verschmutzung miRNA 7 darstellen. Dies wurde vor der Dichtegradient Ultrazentrifugation mit einem im Handel erhältlichen Kits durchgeführt. Für praktische Zwecke ein Drei - Stufen - Standarddichte - Ultrazentrifugation Protokoll , das von kommerziellen Unternehm...

Watch this Scientific Journal Video about Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein at JoVE.com

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

MicroRNAs spielen eine wichtige regulatorische Rolle und werden als neue therapeutische Targets für verschiedene menschliche Krankheiten entstehen. Es hat sich gezeigt, dass miRNAs in Lipoproteinen hoher Dichte durchgeführt werden. Wir haben ein vereinfachtes Verfahren entwickelt, um schnell gereinigter HDL für miRNA-Analyse von Humanplasma geeignet isolieren.

Schnelle und vereinfachte Verfahren für Hochdurchsatz-Isolierung von miRNA aus Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

fast-simplified-method-for-high-through-put-isolation-mirna-from

Research

JoVE Journal

Biology

11.8K Aufrufe.  Virginia Commonwealth University.  MicroRNAs spielen eine wichtige regulatorische Rolle und werden als neue therapeutische Targets für verschiedene menschliche Krankheiten entstehen. Es hat sich gezeigt, dass miRNAs in Lipoproteinen hoher Dichte durchgeführt werden. Wir haben ein vereinfachtes Verfahren entwickelt, um schnell gereinigter HDL für miRNA-Analyse von Humanplasma geeignet isolieren. 

Serie: Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Forschung

Biologie

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantifizierung von dsDNA mit dem Hitachi F-7000 Fluoreszenz-Spektralphotometer und PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute Quantenausbeute Messung von Pulverproben

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Schnelle Fibroblast Entfernung aus High Density humanen embryonalen Stammzellen Kulturen

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Hoch effiziente Ligation kleine RNA-Moleküle für MicroRNA Quantifizierung von Hochdurchsatz-Sequenzierung

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Nachweis von miRNA Targets in Hochdurchsatz-Verwendung des 3&#39;LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolierung von Mitochondrien von minimalen Mengen an Maus-Skelettmuskel für die Hochdurchsatz-Mikroplatten-Atemmessungen

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.