1) Zebrafisch Egg Collection <ol><li> Am Nachmittag vor dem Tag der chemischen Bildschirm einrichten 10 bis 20 Zebrafisch Zuchtbecken. Füllen Sie die einzelnen Behälter mit Wasser aus der Aquakultur System. Mit einem Fangnetz, Transfer ein erwachsenes Männchen und 1-2 erwachsenen Weibchen zu Innenbehälter in jedem Aufzuchtbecken. Separate der männlichen und weiblichen Fische voneinander mit einem Teiler. Beschriften Sie die Käfige und einen Deckel über sie. </li><li> Am Morgen des Bildschirms, entfernen Sie die Teiler von Zuchtbecken und ermöglichen Zebrafisch zu paaren. Im Laufe der nächsten 1 Stunde ermöglichen befruchteten Eizellen durch das Gitter an der Unterseite eines jeden inneren Behälter fallen. </li><li> Nach 1 Stunde zurück Erw. Zebrafisch zurück zur permanenten Speicher, entfernen Sie den inneren Behälter und sammeln die Eier durch Anstrengung das Wasser in jedes Aufzuchtbecken durch eine Kunststoff-Teesieb. </li><li> Kehren Sie die Sieb über einer Petrischale und spülen Sie das Sieb vorsichtig, um die Eier in die Petrischale bündig mit einer Waschflasche mit dem E3 Medium. </li><li> Alle unbefruchteten Eiern, die undurchsichtig erscheinen, sollten entfernt mit einem Einweg-Kunststoff-Pipette werden. Jede Paarung Kreuz sollte Ausbeute etwa 200 Embryonen. </li></ol> 2) Arraying Embryos in 96-well Platten <ol><li> Transfer ca. 5 Embryonen in E3 Medium in jedes Well einer 96-Well-Platte mit einem Glas Pasteur Pipette. </li><li> Einmal Embryonen auf die 96-Well-Platte sind aufgereiht, entfernen Sie so viel von der E3 Medium wie möglich aus dem Brunnen mit einem 12-Kanal (30 bis 300 ul) Pipette, dabei nicht zu durchstechen des Embryos. Mit dem 12-Kanal-Pipette, liefern 250 ul E3-Medium mit Kanamycin 0.5μg/ml in jede Vertiefung so schnell wie möglich, um nicht zu erlauben Embryonen zu versiegen. </li><li> Setzen Sie die 96-Well-Platten in 28,5 ° C Inkubator bis sie die gewünschte Stufe, wenn die Verbindungen hinzugefügt werden zu erreichen. </li></ol> 3) Übertragung von Small Molecule-Bibliothek Während Verbindung Transfer mit Roboter-Transfer-Methoden automatisiert werden können, beschreiben wir die manuelle Methode zur Übertragung. <ol><li> Kleines Molekül-Bibliotheken werden in der Regel in einer 96-Well-Format geliefert, wobei jede Verbindung in DMSO als 10 mM Lager abgelegt. Etwa 60 Minuten vor der Embryonen das Stadium erreicht, wenn die Verbindungen hinzugefügt werden sollen, Tauwetter eine gewünschte Anzahl von 96-well-Platten mit Aliquots von kleinen Molekülen (Quelle Platte). Beachten Sie die Seriennummer oder sonstige Identifizierungsnummer der Quelle Platten. Zur Minimierung von Kondenswasser auf den Platten, kann Auftauen in einer Austrocknung Kammer mit Drierite (WA HAMMOND DRIERITE CO, Xenia, OH) auftreten. </li><li> Kurz-Spin-Down der Platten in einer Tischzentrifuge mit Multi-Well-Platte-Adapter ausgestattet. </li><li> Entfernen Sie die Aluminium-Versiegelung von Quelle Platte. Mit einem 12-Kanal-Pipette, verdünnte die Verbindungen in die Quelle Platte, um die Konzentration von 0,5 mM (zum Beispiel, wenn beginnend mit 250 nL Aliquots von 10mM Lager, fügen 4,75 l DMSO in jede Vertiefung). </li><li> Wenn die Embryonen in der 96-Well-Platte (Empfänger Platte) den gewünschten Zustand zu erreichen, verwenden Sie ein 12-Kanal (2-20 mL) Pipette 2.5μL von Verbindungen (0,5 mm) von der Quelle Platten Transfer in die Empfänger-Platten, die die Embryonen. </li><li> Notieren Sie sich die Kennnummer der Quelle Platten auf den Empfänger Embryo Platten. Decken Sie die Empfänger-Platten nun mit den Embryonen und Verbindungen mit Deckel, vorsichtig mischen die Platten durch vorsichtiges Schwenken, und legen Sie sie in einem 28,5 ° C Inkubator. </li><li> Cover jede Quelle Platte mit ungenutzten kleinen Molekülen (0,5 mM) mit einem Aluminium-Dichtband und in einem -80 ° C Gefrierschrank für langfristige Lagerung. </li></ol> 4) Vorsorgeuntersuchungen für Wirkung von kleinen Molekülen durch visuelle Inspektion der Phänotypen <ol><li> Vor der Durchführung des Bildschirms, formulieren ein bestimmtes Kriterium für das, was wäre ein &quot;Hit&quot; darstellen. </li><li> Am gewünschten Zeiten in der Entwicklung, entfernen Sie die 96-well-Platten, die Verbindung behandelten Embryonen aus Inkubator und prüfen jede Vertiefung unter einem Stereomikroskop. Zur besseren Visualisierung von subtilen Veränderungen, wie zum Beispiel Änderungen in Kreislauf-Muster kann eine Phasen-Kontrast-inverse Mikroskop verwendet werden. Fluoreszenz-Mikroskopie kann verwendet werden, um Störung der Expression von GFP oder DsRed Proteine ​​unter einer Gewebe-spezifischen Promotor zu untersuchen. </li><li> Schnell die 96-Well-Platten für alle gut in die mindestens 3 von 5 Embryonen zeigen die vorgeschriebenen &quot;Hit&quot;-Phänotyp. Notieren Sie sich die Identität der Platte und die auch Standort eines jeden potentiellen Hit. </li><li> Bestätigen Sie ein potentieller Hit von erneute Testen der Auswirkungen der Verbindung bei mehreren Dosen (1 uM, 5 uM, 10 uM und 50 uM). Für jede Dosis wurden 10 Embryonen in 0,5 ml E3-Medien in einer 48-Well-Platte-Format getestet. Der Zeitpunkt der Verbindung hinaus für eine erneute sollte identisch mit dem des ursprünglichen Screening. Ein Hit ist bestätigt, wenn die ausgelöste Phänotyp auf einen erneuten Test der compoun wiedergegebend </li><li> Identifizieren Sie die Hit-Verbindung aus der Datenbank von kleinen Molekülen in der chemischen Bibliothek. </li></ol>

<ol>
	<li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish.">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li><li>Burns, C. G. <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+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors.">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li><li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery/vein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase/ERK+signaling.">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li><li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish.">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li><li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant.">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li><li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation.">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li></ol>

Bei der Planung eines Zebrafisch-basierte chemische Bildschirm, muss besonderes Augenmerk auf die Robustheit der Phänotyp unter Berücksichtigung der Hintergrund von solchen Phänotyp bezahlt werden. Dies ist besonders wichtig für Bildschirme für chemische Suppressoren einer induzierten Phänotyp. Zum Beispiel für einen Phänotyp durch Hitze-Schock-Induktion einer Transgen hervorgerufen, die Bedingung, dass der Phänotyp induziert reproduzierbar muss genau abgebildet werden aus vor Beginn der Bildschirm zu unvertret...

<ol><li> Mindestens 20 Paar erwachsenen Zebrafisch der gewünschten Genotyp. </li><li> Fischernetzen, Zuchtbecken, mit abnehmbarem Innenbehälter und Teiler (aquatische Habitate). </li><li> Petrischalen (10 cm). </li><li> Plastic Teesieb. </li><li> Waschflasche containg Embryo Wasser. </li><li> Einweghandschuhe aus Polyethylen Transferpipetten. </li><li> Polystyrol 96-well Rundboden-Assay-Platten (Corning Costar, Lowell, MA). </li><li> Glass Pasteurpipette (Fisher Scientific). </li><li> Manuelle Pipetten Pumpe, 10 mL (Bel-Art Products, Pequannock, NJ). </li><li> E3 Embryo Medium: 5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl 2, 0,33 mM MgSO 4, mit 0,003% PTU (Phenylthio-Carbamid, Sigma, St. Louis, MO). PTU kann als 10-fach durch Auflösen von 0,3-g PTU in 1 L E3 Embryo Medien vorbereitet werden. Lösungen, die PTU sollten vor Licht durch Abdecken mit Alufolie geschützt werden. </li><li> 12-Kanal-Pipetten, 2-20 ul (Eppendorf). </li><li> 12-Kanal-Pipetten, 3-300 ul (Eppendorf). </li><li> Disposable Pipette Polystyrol-Becken, 50 mL (Fisher Scientific). </li><li> Kleines Molekül-Bibliothek von strukturell unterschiedlichen Verbindungen in einer 96-wll-Format bei 10 mM Lager in DMSO angeordnet. Jeder Master Platte wird in 96-well Polypropylen Speicherplatten (Corning) aliquotiert und bei -80 ° C bis zur Verwendung. </li><li> Aluminium Dichtband für 96-Well-Platten (Nunc, Rochester, NY). </li><li> DMSO (Sigma, St. Louis, MO). </li><li> Grundlegende Inkubator, 28,5 ° C (Fisher Scientific). </li><li> Stereomikroskop mit Durchlichtbasis (Leica Microsystems, Bannockburn, IL). </li></ol>

large scale zebrafish-based in vivo small molecule screen

Aufgrund ihrer kleinen Größe Embryo, schnelle Entwicklung, Transparenz, Fruchtbarkeit und zahlreichen molekularen, morphologischen und physiologischen Ähnlichkeiten zu den Säugetieren, hat Zebrafisch als leistungsfähige entstanden<em&gt; In vivo</em&gt; Plattform für Phänotyp-basierten Wirkstoff-Bildschirme und chemisch-genetischen Analyse. Hier zeigen wir eine einfache, praktische Methode zur groß angelegten Screening von kleinen Molekülen mit Zebrafisch-Embryos.

Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafisch als leistungsfähige entstanden<em&gt; In vivo</em&gt; Plattform für Phänotyp-basierten Wirkstoff-Bildschirme und chemisch-genetischen Analyse. Hier zeigen wir eine einfache, praktische Methode zur groß angelegten Screening von kleinen Molekülen mit Zebrafisch-Embryos.

Large Scale Zebrafisch-Based In-vivo- Small Molecule-Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

large-scale-zebrafish-based-in-vivo-small-molecule-screen

Research

JoVE Journal

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

13.4K Aufrufe.  Vanderbilt University School of Medicine. Zebrafisch als leistungsfähige entstanden In vivo Plattform für Phänotyp-basierten Wirkstoff-Bildschirme und chemisch-genetischen Analyse. Hier zeigen wir eine einfache, praktische Methode zur groß angelegten Screening von kleinen Molekülen mit Zebrafisch-Embryos.

Serie: Large Scale Zebrafish-Based In vivo Small Molecule Screen

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Large-Scale-Bildschirme von Metagenombanken

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Forschung

Biologie

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Batch Immunfärbung für Large-Scale Protein-Nachweis im Whole Affenhirn

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolation und großräumige Ausdehnung der erwachsenen Menschen Endothelial Colony Forming Progenitorzellen

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Small-scale Kernextrakten für funktionelle Assays von Gen-Expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium-Assay für Groß SNP Genotypisierung Anwendungen

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Eine Strategie für einfühlsam, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Ein Handbuch Kleine Molecule Bildschirm Nähern Hochdurch Mit Zebrafischembryonen

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identifizierung neuartiger Regulatoren der Pflanzentranspiration durch großangelegtes Thermisches Bildgebungsscreening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

Ein halbautomatisches ChIP-Seq-Verfahren für groß angelegte epigenetische Studien

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...