Die Finanzierung wurde von der Gordon and Betty Moore Foundation Zuschüsse und der National Science Foundation Grant MCB-0702125 zur Verfügung gestellt.

<ol>
	<li>Liang, P., Pardee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 257 (5072), 967-967 (1992).</li><li>Belasco, J. G., Belasco, J. G., Brawerman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+messenger+RNA+stability.">Control of messenger RNA stability.</a> , 3-11 (1993).</li><li>Poretsky, R. S., Bano, N., Buchan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 71 (7), 4121-4121 (2005).</li><li>Gelder, R. N. V., von Zastrow, M. E., Yool, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Natl. Acad. Sci. USA. 87 (5), 1663-1663 (1990).</li><li>Margulies, M., Egholm, M., Altman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 437 (7057), 376-376 (2005).</li><li>Frias-Lopez, J., Shi, Y., Tyson, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Natl. Acad. Sci. USA. 105 (10), 3805-3805 (2008).</li><li>Poretsky, R. S., Hewson, I., Sun, S., Allen, A. E., Zehr, J. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Environ Microbiol. 11 (6), 1358-1375 (2009).</li></ol>

Die Untersuchung der Genexpression durch natürliche mikrobielle Gemeinschaften hat sich in den letzten Jahren häufig als Mittel, um die ökologische Rolle und Funktion von Mikroorganismen zu entdecken. In Kombination mit dem Nachweis, Quantifizierung und Charakterisierung von marinen Mikroorganismen, können Analysen der Genexpression verwendet werden, um Phylogenie Link in natürlicher mikrobieller Gemeinschaften funktionieren. Viele Techniken der gezielten funktionellen Genexpression beruhen auf spezifischen Sonden ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>PowerSoil Bead Tubes</td>
<td>kit</td>
<td>MoBio</td>
<td>12866-25-PBT</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy Mini Kit</td>
<td>kit</td>
<td>QIAGEN</td>
<td>74104</td>
<td>&nbsp;</td>
<tr>
<td>TURBO DNA-free</td>
<td>kit</td>
<td>Ambion</td>
<td>AM1907</td>
<td>&nbsp;</td>
<tr>
<td>mRNA-ONLY Prokaryotic mRNA Isolation Kit</td>
<td>kit</td>
<td>Epicentre</td>
<td>MOP51010/MOP51024</td>
<td>&nbsp;</td>
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<td>MICROBExpress Bacterial mRNA Enrichment Kit</td>
<td>kit</td>
<td>Ambion</td>
<td>AM1905</td>
<td>&nbsp;</td>
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<td>MICROBEnrich kit</td>
<td>kit</td>
<td>Ambion</td>
<td>AM1901</td>
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<td>MessageAmp II-Bacteria RNA Amplification Kit</td>
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<td>Ambion</td>
<td>AM1790</td>
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<td>Universal RiboClone cDNA Synthesis System</td>
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<td>Promega</td>
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<td>Wizard DNA Clean-Up System</td>
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analyzing gene expression from marine microbial communities using environmental transcriptomics

Analog zu Metagenomik, Umwelt Transkriptomik (metatranscriptomics) abruft und Sequenzen Umwelt mRNAs aus einer mikrobiellen Assemblage ohne vorherige Kenntnis, welche Gene die Gemeinde könnte zum Ausdruck. So bietet die unvoreingenommene Sicht auf Gemeinschaft Genexpression<em&gt; In situ</em&gt;. Environmental Transkriptomik Protokolle sind technisch schwierig, da prokaryotische mRNAs in der Regel die Poly (A)-Enden, die Isolation zu machen von eukaryotischen Nachrichten relativ einfach mangelnde<sup&gt; 1</sup&gt; Und wegen der relativ kurzen Halbwertszeiten von mRNAs<sup&gt; 2</sup&gt;. Darüber hinaus mRNAs viel seltener als rRNAs in Gesamt-RNA extrahiert werden, damit eine rRNA Hintergrund oft überfordert mRNA-Signale. Allerdings haben Techniken zur Überwindung einiger dieser Schwierigkeiten vor kurzem entwickelt worden. Ein Verfahren zur Analyse von Umwelt-Transkriptomen durch die Schaffung von Klon-Bibliotheken unter Verwendung zufälliger Primer Reverse Transkription und verstärken Umwelt mRNAs wurde kürzlich beschrieben, wurde in zwei verschiedenen natürlichen Umgebungen erfolgreich, aber die Ergebnisse waren durch die Wahl des zufälligen Primer verwendet, um cDNA-Synthese zu initiieren voreingenommen<sup&gt; 3</sup&gt;. Advances in lineare Verstärkung der mRNA überflüssig machen random Primer bei der Amplifikation Schritt und machen es möglich, weniger Ausgangsmaterial verwenden Verringerung der Erfassung und Verarbeitung von Proben und minimiert dadurch RNA-Abbau<sup&gt; 4</sup&gt;.<em&gt; In-vitro-</em&gt; Transkription Methoden zur Amplifikation mRNA beinhalten polyadenylating der mRNA wurden, mit einem T7-Promotor auf die 3 Ende des Transkripts. Amplified RNA (aRNA) kann dann auf den cDNA Doppelzimmer mit zufälligen Hexameren und direkt sequenziert von Pyrosequenzierung<sup&gt; 5</sup&gt;. Eine erste Anwendung dieser Methode an der Station ALOHA demonstriert seine Nützlichkeit für die Charakterisierung von mikrobiellen Gemeinschaft Genexpression<sup&gt; 6</sup&gt;.

Watch this Scientific Journal Video about Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics at JoVE.com

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Wir stellen eine Methode zur Erzeugung von cDNA aus Umwelt-mRNA. In der Regel Gesamt-RNA zunächst aus der Umgebung gesammelt, rRNA selektiv entfernt, mRNA wird selektiv verstärkt und cDNA aus der angereicherten mRNA-Pool synthetisiert wird sequenziert. Recovered Sequenzen annotiert werden unter Verwendung von Standard Bioinformatik Techniken, um die exprimierten Gene zu identifizieren.

Analyse von Genexpressionsdaten aus marinen mikrobiellen Gemeinschaften mit Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

analyzing-gene-expression-from-marine-microbial-communities-using

Research

JoVE Journal

Biology

12.4K Aufrufe.  University of Georgia (UGA). Wir stellen eine Methode zur Erzeugung von cDNA aus Umwelt-mRNA. In der Regel Gesamt-RNA zunächst aus der Umgebung gesammelt, rRNA selektiv entfernt, mRNA wird selektiv verstärkt und cDNA aus der angereicherten mRNA-Pool synthetisiert wird sequenziert. Recovered Sequenzen annotiert werden unter Verwendung von Standard Bioinformatik Techniken, um die exprimierten Gene zu identifizieren.

Serie: Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Microbial Communities in Nature and Laboratory - Interview

Mikrobielle Gemeinschaften in Natur und Labor - Interview

Forschung

Biologie

Biology of Microbial Communities  - Interview

Biologie mikrobieller Gemeinschaften - Interview

Bacterial Gene Expression Analysis Using Microarrays

Bakterielle Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Konzentration der Metabolite von Low-Density-Planktonic Gemeinschaften für Umweltforschung Metabolomics mit Kernresonanzspektroskopie

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...