1) Zebra balığı Yumurta Toplama <ol><li> Kimyasal ekran gün önce öğleden sonra, 10 ila 20 Zebra balığı üreme tankları kurdu. Her bir tank su ile yetiştiricilik sistemi doldurun. Bir balık ağı kullanarak, her bir yetiştirme tankında iç konteyner bir yetişkin erkek ve 01:59 yetişkin kadın aktarın. Bir bölücü ile erkek ve dişi balık birbirinden ayırın. Kafesleri Etiket ve üzerlerine bir kapak koydu. </li><li> Ekranın sabah, üreme tankları bölücüler kaldırmak ve çiftleşmek için zebrafish izin. Önceki 1 saat boyunca, döllenmiş yumurta her iç konteyner alt kısmında ızgara üzerinden düşmesine izin vermeyin. </li><li> 1 saat sonra, kalıcı depolama tankları yetişkin zebrafish geri dönüş iç kabı çıkarın ve bir plastik çay süzgeci ile her bir yetiştirme tankında su süzme yumurta toplamak. </li><li> Petri kabı üzerinden süzgeci ters çevirin ve E3 orta içeren bir yıkama şişesi kullanarak Petri kabı içine yumurtaları temizlemek için yavaşça süzgeç durulayın. </li><li> Opak görünür tüm döllenmemiş yumurta, tek bir plastik pipet kullanarak çıkarılmalıdır. Her çiftleşme çapraz yaklaşık 200 embriyolar verim gerekir. </li></ol> 2) 96-kuyu Tabaklar Embriyolar Arraying <ol><li> 5, 96 plaka bir cam Pasteur pipeti kullanarak her bir kuyunun içine E3 ortamda embriyolar hakkında aktarın. </li><li> Embriyo ponksiyon bakım alarak, pipet embriyolar 96-plaka üzerine dizilmiş, bir 12-kanal (300 mcL 30) kullanarak kuyuların mümkün olabildiğince kadar E3 orta kaldırmak. 12-kanallı pipet kullanarak, böylece embriyolar kadar kuruması için izin vermemek için de mümkün olduğunca çabuk, her 0.5μg/ml kanamisin içeren E3 orta 250 mcL sunmak. </li><li> 96-kuyucuğu bileşikler eklenecek zaman istenilen aşamaya ulaşmak kadar 28.5 ° C inkübatör içine koyun. </li></ol> 3) Küçük Molekül Kütüphanesi Devri Bileşik transferi robotik transfer yöntemleri ile otomatik olabileceği gibi, biz manuel aktarma yöntemini anlatacağız. <ol><li> Küçük molekül kütüphaneleri, tipik olarak 10 mM stok olarak DMSO saklanan her bileşik ile, 96-well formatında temin edilmektedir. Bileşikler eklenecek embriyolar aşamaya ulaşmak yaklaşık 60 dakika önce, küçük moleküller aynı hacimde (kaynak plaka) içeren 96-kuyucuğu İstediğiniz bir numarayı Çözülme. Kaynak plakaların seri veya diğer kimlik numarası dikkate alınız. Plakalar üzerinde yoğunlaşma en aza indirmek için, buz eritme Drierite (WA HAMMOND DRIERITE CO, Xenia, OH) içeren bir kuruma odasında oluşabilir. </li><li> Kısaca çok plaka adaptörü ile donatılmış bir masa üstü santrifüj plakaları dönerler. </li><li> Kaynak plaka alüminyum sızdırmazlık bantı çıkarın. 12-kanallı pipet kullanarak, kaynak plaka bileşiklerin konsantrasyonu 0.5 mM (örneğin, 250 nL alikotları 10mM stok ile başlayan, her bir kuyunun 4.75 mcL DMSO eklemek) seyreltin. </li><li> 96-plaka embriyoların (alıcı plaka) istenilen aşamaya ulaşmak, alıcı plakalar içeren kaynak plakaları bileşiklerin 2.5μL (0.5mm) aktarmak için bir 12-kanal (2-20 mcL) pipet kullanın embriyoların. </li><li> Alıcı embriyo plakalar üzerinde kaynak plakalarının kimlik numarasını kaydedin. Kapak kapakları ile embriyoların ve bileşikleri içeren alıcı plakaları, yavaşça yavaşça dönen plakalar karışımı, ve 28.5 ° C inkübatör koyun. </li><li> Uzun süreli saklama için -80 ° C derin dondurucuda alüminyum sızdırmazlık bantı ve yer ile kullanılmayan küçük moleküller (0.5 mM) içeren her kaynak plaka Kapak. </li></ol> 4) Küçük Moleküllerin Etkileri fenotipleri Görsel Muayene Tarama <ol><li> Ekranın gerçekleştirmeden önce, ne bir &quot;hit&quot; teşkil edeceğini için belirli bir kriter formüle. </li><li> Gelişimi istenilen zamanlarda, inkübatör bileşik tedavi edilen embriyolar içeren 96-kuyucuğu çıkarın ve bir stereomikroskop altında her iyi incelemek. Dolaşım desen değişiklikleri gibi ince değişiklikler, daha iyi bir görünüm için, ters faz-kontrast mikroskop kullanılabilir. Floresan mikroskobu, bir doku spesifik organizatörü altında GFP veya DsRed proteinleri ifade pertürbasyon incelemek için kullanılabilir. </li><li> 5 embriyo en az 3 öngörülen &quot;hit&quot; fenotip sergileyen herhangi bir kuyu için 96-kuyucuğu hızla tarayın. Kimlik, plaka ve her potansiyel hit iyi bir konuma kaydedin. </li><li> Çeşitli dozlarda (1 UM, 5 UM, 10 mcM ve 50 mcM) bileşik etkileri yeniden test potansiyel bir hit teyit. Her doz için 48 plaka formatında E3 medya 0.5 ml, 10 embriyolar test edilmektedir. Test için bileşik Ayrıca zamanlama orijinal tarama aynı olmalıdır. Hit ortaya çıkardı fenotip compoun, yeniden test yeniden teyitd </li><li> Küçük moleküllerin kimyasal kütüphane veritabanı isabet bileşik belirleyin. </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>

Zebrafish bazlı kimyasal ekran planlama yaparken özellikle dikkat sağlamlık fenotip ve arka plan gibi fenotip oranı dikkate alınarak ödenmelidir. Bu kimyasal bastırıcılar kaynaklı bir fenotip ekranlar için özellikle önemlidir. Örneğin, bir transgen ısı-şok indüksiyon kaynaklanan bir fenotip, fenotipe neden olan durum tekrarlanabilir kabul edilemeyecek kadar yüksek yalancı pozitiflik oranları önlemek için tam ekran başlamadan önce eşlenmiş olmalıdır. Dikkatli bir planlama ile, mütevazı bi...

<ol><li> En az 20 çift istenilen genotip yetişkin zebrafish. </li><li> Balık ağları, Hayvancılık tankları, çıkarılabilir iç konteyner ve bölücüler (Aquatic Habitat). </li><li> Petri kapları (10 cm). </li><li> Plastik çay süzgeci. </li><li> Şişe containg embriyo su yıkayın. </li><li> Tek kullanımlık polietilen transferi pipetler. </li><li> Polistiren 96-iyi yuvarlak alt test plakaları (Corning Costar Lowell, MA). </li><li> Cam Pasteur pipeti (Fisher Scientific). </li><li> Manuel pipet pompası, 10 ml (Bel-Sanat Ürünleri, Pequannock, NJ). </li><li> E3 embriyo orta:% 0.003 PTU (phenylthio-karbamit, Sigma, St Louis, MO) içeren 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2, 0.33 mM MgSO 4,. PTU 1 L E3 embriyo medya 0.3 g PTU eriterek 10x bir çözüm olarak hazırlanabilir. PTU içeren Çözümleri alüminyum folyo ile kaplayarak ışıktan korunmalıdır. </li><li> 12-kanallı pipetler, 2-20 mcL (Eppendorf). </li><li> 12-kanallı pipetler, 3-300 mcL (Eppendorf). </li><li> Tek kullanımlık polistiren pipet havzası, 50 ml (Fisher Scientific). </li><li> DMSO 10 mM stok az 96 wll formatında dizilmiş yapısal olarak farklı bileşiklerin küçük molekül kütüphanesi. Her ana plaka 96-iyi polipropilen depolama levhalar (Corning) aliquoted ve -80 ° C kullanana kadar saklanır. </li><li> Alüminyum (Nunc, Rochester, NY) 96-kuyucuğu için bant mühürleme. </li><li> DMSO (Sigma, St Louis, MO). </li><li> Temel inkübatör, 28.5 ° C (Fisher Scientific). </li><li> Iletilen ışık tabanı (Leica Microsystems, Bannockburn, IL) ile stereomikroskopta. </li></ol>

large scale zebrafish-based in vivo small molecule screen

, Küçük embriyo boyutu, hızlı bir gelişme, şeffaflık, doğurganlık ve memeliler için çok sayıda moleküler, morfolojik ve fizyolojik benzerlikleri göz önüne alındığında, zebrafish güçlü olarak ortaya çıkmıştır<em&gt; In vivo</em&gt; Fenotip tabanlı ilaç ekranları kimyasal ve genetik analiz platformu. Burada, küçük moleküller zebrafish embriyoları kullanılarak büyük ölçekli tarama için basit, pratik bir yöntem gösteriyor.

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

Zebra balığı güçlü olarak ortaya çıkmıştır<em&gt; In vivo</em&gt; Fenotip tabanlı ilaç ekranları kimyasal ve genetik analiz platformu. Burada, küçük moleküller zebrafish embriyoları kullanılarak büyük ölçekli tarama için basit, pratik bir yöntem gösteriyor.

Büyük Ölçekli Zebra balığı Tabanlı In vivo Küçük Molekül Ekran

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

13.4K Görüntüleme.  Vanderbilt University School of Medicine. Zebra balığı güçlü olarak ortaya çıkmıştır In vivo Fenotip tabanlı ilaç ekranları kimyasal ve genetik analiz platformu. Burada, küçük moleküller zebrafish embriyoları kullanılarak büyük ölçekli tarama için basit, pratik bir yöntem gösteriyor.

Video: 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.

Metagenomic Kütüphaneler Büyük Ölçekli Ekranlar

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

Biyoloji

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.

Tüm Maymun Beyin Büyük Ölçekli Protein Algılama için Toplu immün

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.

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

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.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

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.

Gen-ifadesi Makinaları Fonksiyonel Tahliller için Küçük ölçekli Nükleer ayıklar

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.

Büyük ölçekli SNP Genotiplendirme Uygulamaları için infinium Tahlili

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.

Hassas, Büyük Ölçekli Kantitatif metabolomik için bir strateji

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.

Zebra balığı Embriyolar kullanılarak Yüksek verim Yaklaşıyor A Manuel Küçük Molekül Ekran

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

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

Probiyotik Fermente Gıdaların küçük ölçekli imalat için yeni Üretim Protokolü

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Maya Büyümesinin Çok Parametreli Niceleşmesi İçin Büyük Ölçekli Bir Yöntem

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...