Name: simple bulk readout of digital nucleic acid quantification assays
Uploaded: 2015-09-24T15:00:00
Description: Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

Remarque: Fabrication du dispositif microfluidique est pas nécessaire pour cet essai, sous forme de gouttelettes de ce protocole peuvent être formés avec des décideurs de gouttelettes commerciales existantes 23,29. 1. Faire le dispositif microfluidique gouttelettes formant <ol><li> Préparer moule maître pour les canaux avec le protocole SU-8 Fabrication de Maître indiqué précédemment 31, mais avec un masque motif générateur de gouttelettes 32.<...

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Essais numériques sont puissantes méthodes qui permettent la détection de cellules rares et de comptage individuels des molécules d&#39;acide nucléique. Cependant, les essais numériques sont toujours pas appliquées systématiquement dans les laboratoires d&#39;analyse, en partie à cause du coût de l&#39;équipement spécialisé associée à des méthodes disponibles dans le commerce. Nous décrivons ici une analyse numérique pour quantifier extrémité des acides nucléiques avec un affichage analogique simpli...

Essais numériques pour la quantification de l&#39;acide nucléique (PCR numérique) 1-4 et la base afin (séquençage) sont fortement un impact sur ​​les sciences de la vie et de la médecine. Dosages numériques offrent quantification des chiffres moléculaires sur une échelle absolue (pas par rapport à un contrôle), offrant une haute sensibilité, permettant des comparaisons faciles à travers des expériences, et, surtout, permettant la construction de grandes bases de données contenant des données comparables 5 (Tableau 1). Au cours des 15 dernières années, toute amplification du génome (WGA) est apparu aux côtés de PCR comme un outil général pour l&#39;amplification de l&#39;acide nucléique. Comme PCR, WGA est utile pour les applications d&#39;analyse et de préparation en amplifiant éléments de l&#39;échantillon minute jusqu&#39;à un niveau qui peut être facilement détecté ou utilisé pour des analyses ultérieures comme le séquençage de base. Contrairement à la PCR, WGA sont pas spécifiques à un locus d&#39;ADN particulière, ce qui permet plutôt amplification de toutes les séquences de l&#39;échantillon, y compris des séquences inconnues.Cette différence fondamentale entre PCR et WGA rend les méthodes complémentaires les uns aux autres et donnent lieu à des problèmes différents dans leur application. Le rendement élevé de réactions WGA 6 permet l&#39;amplification de routine de l&#39;ADN génomique à partir de molécules simples 7, 8 cellules individuelles et d&#39;autres échantillons à faible biomasse 9 pour la quantification ou une analyse plus approfondie. Les principaux défis associés à la chimie WGA sont son extrême sensibilité aux contaminants et l&#39;amplification inégale entre les molécules de matrice individuelles 6. Popularité Cependant, WGA gagne que le séquençage cellule unique a émergé comme le &quot;killer application&quot; de la technologie de WGA 10, et le modèle quantification par WGA est important dans de nombreux domaines d&#39;application 7. Instrumentation pour la quantification d&#39;acide nucléique numérique a été précédemment décrit dans une variété de valved et sans valve microfluidique forma11-14 ts, y compris les essais à base de gouttelettes 15,16 (tableau 2). Cependant, les systèmes microfluidiques commerciales pour l&#39;analyse numérique nécessitent de l&#39;équipement spécialisé pour la configuration de réaction et la détection de produits 17. Microfluidique valved personnalisée sont flexibles, mais nécessitent microfabrication de précision et des systèmes de contrôle pneumatiques 18. Alors qu&#39;il est relativement simple de faire des micro-gouttelettes monodispersées pour des essais numériques à base d&#39;émulsion 19,20, affichage numérique est techniquement lourde, nécessitant soit l&#39;imagerie à grand champ à grande échelle (similaire aux technologies populaires de séquençage de prochaine génération) ou 21,22 haute vitesse d&#39;écoulement à base de détection de gouttelette 23-25. Idéalement, une analyse numérique serait simple de mettre en place à la lecture, réduisant la nécessité d&#39;une instrumentation complexe et permettant un grand nombre d&#39;échantillons à lire rapidement. Nous décrivons ici une méthode simplifiée pour la lecture des tests de gouttelettes numériques qui utilise en temps réel classique PCR iNSTRUMENT pour mesurer la fluorescence en vrac de tests numériques de gouttelettes d&#39;émulsion. Alors que la nouvelle approche peut être appliquée à des tests de PCR numériques, il est particulièrement avantageux pour les dosages WGA numériques analogiques pour lesquels les tests d&#39;amplification en temps réel (qui donnent d&#39;excellents résultats pour la PCR) sont problématiques. WGA est généralement appliquée à des échantillons de molécules de matrice qui sont hétérogènes en séquence, la longueur et le contenu de la base. Ces différentes molécules de matrice sont amplifiés à des taux différents 6, ce qui nécessite l&#39;utilisation d&#39;une référence (&quot;standard&quot;) avec des caractéristiques correspondant à l&#39;échantillon. Souvent, de telles normes est disponible, ou les caractéristiques des échantillons entrants sont inconnus. L&#39;hétérogénéité de la matière d&#39;entrée et des biens dépendant de la longueur des chimies WGA 26 compliquent également l&#39;interprétation des résultats par la création d&#39;ambiguïté dans ce qui est quantifié - la masse d&#39;entrée, le numéro d&#39;entrée de molécules, une combinaison des deux, ou aucun des deux. Fnfin, la séquence de non-spécificité rend quantitative amplification par déplacement multiple (MDA) plus sensibles à la contamination que la PCR quantitative puisque les molécules contaminantes de toute séquence ont un potentiel d&#39;interférer. Dosages numériques microfluidiques lutter contre la contamination en séparant les molécules de modèle et de la réduction des volumes de réaction de telle sorte que moins de contaminants sont échantillonnés. Ici, nous utilisons une méthode WGA isotherme populaire, MDA 27. Fait à noter, plusieurs autres chimies WGA y compris PicoPlex et malbac 28 dépendent de façon cruciale sur les mesures initiales de déplacement isotherme de brins. Étapes isothermes exacerbent le défi de l&#39;application de tests analogiques en temps réel pour WGA quantitative. WGA ne peut ni être entièrement évitée pendant l&#39;installation, conduisant à indésirables pré-amplification variable, ni discrétisation (&quot;cycling&quot;) dans une voie où la réplication de molécules hétérogènes peut être conduit à son terme et a cessé avant le prochain cycle comme PCR 11 </sup&gt; (Figure 1). Un format numérique de dosage pour WGA accueille procédures typiques de configuration de réaction due à la ségrégation de chaque molécule pour le dénombrement au point final de dosage (sorte de pré-amplification n&#39;a pas d&#39;incidence sur les résultats) lecture originale signifie précision serait largement indépendante de la variation de l&#39;efficacité d&#39;amplification. Le dosage dépend de gouttelettes produites dans l&#39;huile avec des volumes uniformes, que le niveau du signal par gouttelette à l&#39;extrémité de dosage dépendra du volume de gouttelettes, et nous ne voulons pas la cohérence des résultats de dépendre d&#39;une moyenne à travers une distribution de tailles de gouttelettes. Faire gouttelettes monodispersées est maintenant une procédure standard (environ 3.500 documents de gouttelettes monodispersées ont été publiés depuis 2013), mais nécessite une instrumentation microfluidique 25. En fait, plus de six sociétés ont développé des produits commerciaux indépendants qui comptent sur la production de ces gouttelettes, et puces microfluidiques gouttelettes de décision sont23,29 disponible dans le commerce. Pour cette étude, nous avons utilisé des dispositifs microfluidiques personnalisé produites en interne (voir Protocole). Pompes seringue pour conduire l&#39;écoulement à travers les dispositifs sont également disponibles dans le commerce, mais encore, peut être remplacé par une seule seringue jetable pour l&#39;écoulement entraîné sous vide pour réduire les coûts 30.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Evagreen Dye</td>
			<td>Biotium</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>New England Biotechnologies</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi29 DNA Polymerase Reaction Buffer</td>
			<td>New England Biotechnologies</td>
			<td>B0269S</td>
			<td>Vortex periodically until aggregate dissolves</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England Biotechnologies</td>
			<td>N3011S</td>
			<td>Heated to 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m in Stratalinker 2400 before use (optional)</td>
		</tr>
		<tr>
			<td>ROX reference dye</td>
			<td>Life Technologies</td>
			<td>12223-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR optical strip caps</td>
			<td>Life Technologies</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP (25uM each)</td>
			<td>Agilent Technologies</td>
			<td>200415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler - Mx3000P qPCR system</td>
			<td>Agilent Technologies</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier pipette tips</td>
			<td>VWR</td>
			<td>89003-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-rad oil for evagreen</td>
			<td>Bio-rad</td>
			<td>186-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>JumpStart Taq ReadyMix</td>
			<td>Sigma-Aldrich</td>
			<td>P2893-100RXN</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple bulk readout of digital nucleic acid quantification assays

Essais numériques sont puissantes méthodes qui permettent la détection de cellules rares et de comptage individuels des molécules d&#39;acide nucléique. Cependant, des essais numériques sont toujours pas systématiquement appliqué, en raison du coût et des équipements spécifiques associés aux méthodes disponibles dans le commerce. Nous présentons ici une méthode simplifiée pour la lecture des tests de gouttelettes numériques en utilisant un instrument conventionnel-PCR en temps réel pour mesurer la fluorescence en vrac de tests numériques de gouttelettes d&#39;émulsion. Nous caractérisons les performances de l&#39;essai de lecture en vrac en utilisant des mélanges de gouttelettes de synthèse et une analyse numérique des gouttelettes amplification par déplacement multiple (MDA). Quantitative MDA particulier bénéficie d&#39;un format numérique, mais la réaction de notre nouvelle méthode applique à toute analyse numérique. Pour les protocoles d&#39;essai numériques établies telles que la PCR numérique, cette méthode sert à accélérer et simplifier dosage lecture. Notre méthodologie vrac de lecture apporte les avantages de partitioneessais D, sans la nécessité d&#39;une instrumentation spécialisée lecture. Les principales limites de la méthodologie de lecture encombrement sont réduits gamme dynamique par rapport aux plates-formes de gouttelettes de comptage et la nécessité d&#39;un échantillon standard, bien que les exigences de cette norme sont moins exigeants que pour une expérience en temps réel classique. Quantitative toute amplification du génome (WGA) est utilisé pour tester les contaminants dans les réactions WGA et est le moyen le plus sensible pour détecter la présence de fragments d&#39;ADN avec des séquences inconnues, donnant la méthode très prometteuse dans divers domaines d&#39;application, y compris le contrôle de la qualité pharmaceutique et l&#39;astrobiologie.

Alors que les lectures en vrac classique / en temps réel peuvent être utilisés à la fois pour la PCR quantitative et analyses quantitatives de WGA (Figure 1), des essais quantitatifs numériques offrent des avantages (tableau 1). Dans le procédé décrit, nous lisons des dosages numériques au format micro-gouttelettes avec une mesure de point final en vrac simple (Figure 2). Bien que cette méthode est largement applicable, nous nous concentrons sur WGA quantitati...

Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Lecture simple de vrac numériques acide nucléique quantification dosages

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

simple-bulk-readout-of-digital-nucleic-acid-quantification-assays

Research

JoVE Journal

Biology

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolume quantification des acides nucléiques

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Recherche

Biologie

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Electricité-libre, l&#39;acide nucléique et séquentielle isolement des protéines

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

D&#39;analyser et de structures d&#39;acides nucléiques avec 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Préparation, imagerie, et la quantification de la surface bactérienne motilité dosages

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

L&#39;extraction d&#39;acide nucléique et efficace de l&#39;ARNr 16S bactérien pour Séquençage génique communautaire Caractérisation

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolement des acides nucléiques: Une méthode d&#39;extraction de l&#39;ADN simple et rapide

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Le test de fixation du Lactate déshydrogénase — Une méthode Simple et fiable pour déterminer l’activité de séquestration autophagique en vrac dans des cellules de mammifères

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Scrunching planaire comme une lecture comportementale quantitative pour la détection de stimuli nocifs

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Protocoles de PCR numérique en gouttelettes à transcription inverse en deux étapes pour la détection et la quantification du SRAS-CoV-2

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...