Gli autori ringraziano il prof. Chiara Campanella e Centro di Competenza AMRA, Università di Napoli Federico II, Napoli, Italia, per fornire loro l&#39;uso del microdissector laser, e il signor Vincenzo Vicidomini per il generoso aiuto per l&#39;animazione 3D.

Parte 1. Preparazione di Drosophila Dischi Ala immaginale Sottoposto a specifico e localizzato RNAi. Come materiale biologico, abbiamo usato Drosophila dischi ala immaginale ottenuti da larve transgeniche sottoposto al silenziamento del gene localizzato e specifico per mezzo del sistema GAL4/UAS 1. Questa progenie larvale orginates da un incrocio genetico che coinvolge due linee parentali: uno porta la conducente GAL4, il secondo il risponditore SUP. Oltre ad esprimere GAL4 nel modello temporale specifico e / o spaziali, la linea pilota porta una SUP-GFP giornalista che permette di visualizzare il dominio GAL4 espressione. La linea UAS risponditore esprime, sotto il controllo della sequenza UAS, un tornante silenziamento dell&#39;RNA (hpRNA) in grado di indirizzare specificamente il gene di interesse selezionati (lo chiamano gene X) 2. Una volta espressa nella cellula, l&#39;X è hpRNA spaccati di generare piccoli RNA interferenti (siRNA) in grado di mettere a tacere specificamente il gene selezionato. Nella progenie larvale, che porta entrambi i driver e risponditore transgeni, le scuole universitarie professionali di silenziamento costrutto è solo trascritto in quelle cellule che esprimono la proteina GAL4, ed è quindi in grado di indurre un silenziamento spazialmente limitato del gene X. selezionato Tra le varietà di possibili applicazioni, abbiamo applicato questo metodo per silenziare un gene selezionato di nostro interesse (gene X) sotto il controllo del engrailed-GAL4 (it) driver, che può innescare silenziamento specifici nel vano posteriore del disco ala 3 , il cui territorio è stato caratterizzato da l&#39;espressione del transgene UAS-GFP. I dischi silenziati ala immaginale sono stati sezionati a mano, avendo cura di eliminare i tessuti circostanti contaminanti, soprattutto i tracheas aderenti. Ogni disco ala isolato è stato poi rapidamente e delicatamente trasferito in un precedentemente trattati, telaio dissezione RNasi gratuito per microdissezione laser immediata di aree selezionate. Parte 2. Microdissezione laser delle aree selezionate dal silenzio (posteriore) e unsilenced (anteriore) comparti del disco ala. Una volta che il disco ala era sulla membrana, microdissezione laser delle aree specifiche di tacere (posteriore; GFP-etichetta) e unsilenced (anteriore; GFP-senza etichetta) i compartimenti è stato raggiunto. Questo disegno è stato eseguito da una zona che potrebbe essere facilmente inserito in entrambi i lati GFP-positive e GFP-negativi del disco. Sotto la microdissector, per prima cosa focalizzato il fascio laser sul tessuto GFP-positive, effettuando un taglio lungo il perimetro selezionato. Il ricavato microdissector con taglio alla velocità selezionata e potere, ma è possibile affinare il taglio manuale, fino a quando l&#39;area selezionata del tessuto cade nel tubo sottostante. Questo tubo contiene un piccolo volume di TRI Reagent, in modo che la GFP-positive tessuto è pronto per la successiva estrazione di RNA. Successivamente abbiamo spostato la zona di taglio per il contrario, GFP-negativi lato del disco ala, al fine di analizzare un territorio equivalente dal unsilenced, GFP-negativi tessuti. Ancora una volta, dopo il taglio automatico, si è proceduto a perfezionare il taglio manualmente. Una volta che il taglio è stato fatto, anche la GFP-negativa del tessuto è stato recuperato nelle TRI Reagent, pronto per l&#39;estrazione di RNA. Parte 3. RNA Estrazione e Profiling Trascrizione da aree di tessuto microdissezionate. Abbiamo girato per staccare i tubi del liquido dal tappo e ha aggiunto TRI Reagent fino a 1 ml, quindi eseguita l&#39;estrazione di RNA seguendo il protocollo standard di TRI Reagent. Per raccogliere materiale sufficiente, abbiamo ripetuto la procedura di taglio sui 100 dischi ala immaginale. A partire da 100 aree di circa 5000 micron 2 ciascuno, il nostro rendimento è stato di 1,5 mg di RNA. Precisione della dissezione manuale è stato poi controllato controllando espressione GFP nei campioni di tacere e unsilenced mediante RT-PCR, utilizzando Super Script III (Invitrogen) per sintetizzare cDNA con esameri casuali. Successive analisi quantitative focalizzate per determinare i livelli di espressione del gene X a tacere, insieme a quelle di target putativi del suo percorso normativo, sono state eseguite da Real Time RT-PCR utilizzando Master Mix (Invitrogen).

<ol>
	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

Per quanto riguarda i loro livelli di espressione basale raggiunti negli scompartimenti anteriore / posteriore dei dischi ala unsilenced, l&#39;attività del gene X selezionato è stato trovato ridotto al 40% alla tacere. Al contrario, uno dei suoi obiettivi putativi (geni Y) è stata trovata significativamente up-regolati (7 volte-aumento), che ci porta a convalidare l&#39;ipotesi che fosse regolato negativamente dal gene X. Concludiamo che l&#39;approccio sperimentale sopra descritto può ...

Vola ceppi Drosophila UAS-silenziamento linee possono essere ottenute da VDRC RNAi collezione 2. Una collezione di GAL4 linee-guida è disponibile presso il Centro archivi Drosophila Bloomington (Indiana, USA). Attrezzatura Attrezzatura richiesta comprende microdissector laser (Leica LMD6000), Coulter Microcentrifugare 22R (Beckman), PCR (BIO-RAD mio Cycler), Real Time PCR (BIO-RAD iQ5). Strumenti Strumenti necessari sono: vetro pozzi, spazzola, pinzette microdissezione, scivoli goccia, spille insetto montati su aghi da siringa, telai in metallo con membrana in PET, tubi di plastica (50 ml, 05 ml, 0,25 ml).

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

Natura eterogenea dei tessuti ha dimostrato di essere un fattore limitante per la quantità di informazioni che possono essere generati da campioni biologici, compromettendo le analisi a valle. Considerando le associazioni complesso e dinamico cellulari esistenti in molti tessuti, per ricapitolare le interazioni in vivo analisi molecolare si deve essere in grado di analizzare popolazioni di cellule specifiche all&#39;interno del loro contesto d&#39;origine. Laser-mediata microdissezione può raggiungere questo obiettivo, consentendo l&#39;identificazione univoca e la raccolta di successo delle cellule di interesse sotto il diretto visualizzazione microscopica, pur mantenendo l&#39;integrità molecolare. Abbiamo applicato questa tecnologia per analizzare l&#39;espressione genica nelle aree definite di sviluppare disco Drosophila ala, che rappresenta un vantaggioso sistema modello per studiare il controllo della crescita, differenziazione cellulare e l&#39;organogenesi. Larvale dischi immaginale precocemente sono suddivise in compartimenti anteriore e posteriore, dorsale e ventrale da confini restrizione lignaggio. Avvalendosi della inducibile GAL4-UAS sistema di espressione binario, ciascuno di questi comparti possono essere etichettate in modo specifico le mosche transgeniche esprimere un transgene UAS-GFP sotto il controllo del competente GAL4-driver costruire. Nei dischi transgenici, il profilo di espressione genica di sottoinsiemi discreti di cellule possono essere, appunto, determinata dopo microdissezione laser-mediata, utilizzando il segnale fluorescente GFP guidare taglio laser. Tra le varietà di applicazioni a valle, ci siamo concentrati sulla profilazione trascrizione RNA dopo localizzato interferenza dell&#39;RNA (RNAi). Con l&#39;avvento della tecnologia RNAi, GFP etichettatura può essere accoppiato con knockdown localizzata di un dato gene, che consente di determinare la risposta trascrizionale di una popolazione di cellule discreti per il silenziamento genico specifico. Per convalidare questo approccio, abbiamo sezionato aree equivalenti del disco dalla parte posteriore (l&#39;etichetta di espressione GFP), e la parte anteriore (non marcato) su vano tacere regionali nel comparto P di un altro gene ubiquitariamente espresso. L&#39;RNA è stato estratto da microdissezionate aree tacere e unsilenced e comparativa profili di espressione genica determinata dalla quantitativa real-time RT-PCR. Abbiamo dimostrato che questo metodo può essere efficacemente applicato per trascrittomica accurata di sottoinsiemi di cellule all&#39;interno dei dischi immaginale Drosophila. Infatti, mentre la preparazione del disco di massa come fonte di RNA assume generalmente omogeneità cellulare, è ben noto che l&#39;espressione trascrizionale può variare notevolmente all&#39;interno di queste strutture a seguito di informazioni di posizione. Utilizzando localizzato segnale fluorescente GFP per guidare taglio laser, analisi trascrizionale più accurata può essere eseguita e proficuamente applicati alle domande più disparate, tra cui profili trascrizione di linee cellulari distinte nel loro contesto d&#39;origine.

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Microdissezione laser è stato applicato per analizzare il profilo di espressione genica in specifici comparti di disco Drosophila ala sottoposta a localizzate RNAi<em&gt; In vivo</em&gt;. RNA estratto da aree equivalenti dei comparti tacere e unsilenced è stato analizzato mediante RT-PCR quantitativa per determinare comparativa profili di espressione genica nel contesto di microecology tessuto nativo.

Microdissezione laser applicata a profili di espressione genica di sottogruppo di cellule dal Drosophila Ala Disco

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

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Research

JoVE Journal

Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

11.1K Views.  University of Naples. Microdissezione laser è stato applicato per analizzare il profilo di espressione genica in specifici comparti di disco Drosophila ala sottoposta a localizzate RNAi In vivo. RNA estratto da aree equivalenti dei comparti tacere e unsilenced è stato analizzato mediante RT-PCR quantitativa per determinare comparativa profili di espressione genica nel contesto di microecology tessuto nativo.

Video: Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser Microdissezione Cattura di tessuti di mammiferi

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Ricerca

Biologia

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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

L&#39;isolamento a base di affinità di Tagged Nuclei di<em&gt; Drosophila</em&gt; tessuti per Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.