Yazarlar prof teşekkür ederim. Chiara Campanella ve Amra Yetkinlik Merkezi, lazer microdissector kullanımını sağlayarak Naples Federico II Üniversitesi, Napoli, İtalya, ve 3D animasyon cömert yardım için Bay Vincenzo Vicidomini.

Bölüm 1. Özgül ve Lokalize RNAi Maruz Drosophila hayali Kanat Diskler hazırlanması. Biyolojik madde olarak, elde edilen transgenik larvaları GAL4/UAS sistemi 1 yerelleştirilmiş ve özel gen susturulması tabi Drosophila hayali kanat diskler kullanılır. GAL4 sürücü, ikinci UAS cevaplayıcı taşıyan: Bu larva döl iki ebeveyn hatları içeren bir genetik çapraz orginates. Belirli bir zamansal ve / veya uzaysal desen GAL4 ifade etmek için ek olarak, sürücü hattı GAL4 ifade etki alanı görüntülemenizi sağlar UAS-GFP muhabiri taşır. UAS cevaplayıcı hattı, UAS sırası kontrolü altında bir saç tokası, susturma özellikle ilgi seçilen gen (çağrı geni X) 2 hedef RNA (hpRNA) ifade eder. Hücrede ifade hpRNA X oluşturmak için özel olarak seçilen gen susturmak için küçük bir müdahale RNA (siRNA) bölünmüş. Larva döl, sürücü ve UAS susturulması inşa GAL4 protein ifade bu hücrelerin sadece transkripsiyonu cevaplayıcı transgenlerin taşır ve böylece, seçilen gen X mekansal sınırlı susturulması uyarabildiklerini Çeşitli olası uygulamalar arasında, engrailed-GAL4 kontrolü altında bizim ilgi seçilen bir gen (gen X) (tr) sürücüsü, kanat disk 3 posterior kompartman belirli susturulması tetikleyebilir susturmak için uygulanan bu yaklaşım , topraklarından UAS-GFP transgen ifadesi damgasını vurdu. Susturuldu hayali kanat diskler, el disseke özellikle çevre kirlenmesine neden olan dokular, yapıştırma tracheas ortadan kaldırmak için özen. Her izole kanat disk sonra hızlı bir şekilde ve yavaşça seçilen alanların hemen lazer mikrodiseksiyon için önceden tedavi, RNaz ücretsiz diseksiyonu çerçeve nakledildi. Bölüm 2. Lazer mikrodiseksiyon kanat disk susturuldu (posterior) ve (ön) unsilenced bölmeleri seçilmiş alanlarda. Unsilenced (anterior; GFP-etiketlenmemiş) bölmeleri elde edildi; sonra kanat disk susturuldu (GFP etiketli arka) belirli alanlarda lazer mikrodiseksiyon, membran üzerinde oldu. Bu bir alana çekerek, hem de kolayca sığabilir yapıldı, disk GFP pozitif ve GFP-negatif tarafları. Microdissector altında, ilk seçilen çevresince bir kesim, lazer ışını GFP pozitif doku üzerinde duruldu. Microdissector seçilen hız ve güç kesme ile devam eder, ancak doku seçilen alanın altındaki tüp düşene kadar, elle kesim rafine için mümkündür. Bu tüp GFP pozitif doku sonraki RNA ekstraksiyonu için hazır olması, TRI Reaktif küçük bir hacim içerir. Biz sonradan unsilenced, GFP-negatif doku eşdeğer bir bölgeyi incelemek amacıyla, GFP-olumsuz yan kanat disk, tam tersi kesim alana taşındı. Bir kez daha, sonra otomatik kesme, el kesme rafine başladı. Sonra RNA ekstraksiyonu için hazır kesilmiş, GFP-negatif doku TRI Reaktif kurtarıldı, yapıldı. Bölüm 3. Microdissected Doku Alanları RNA Ekstraksiyon ve Transkripsiyon Profilleme. Biz kap sıvı ayırmak için tüpler döndü ve daha sonra standart TRI Reaktif protokolü takip RNA ekstraksiyonu, 1 ml TRI Reaktif eklendi. Yeterince malzeme toplamak için, 100 hayali kanat diskler üzerinde kesme işlemi tekrarlanır. Yaklaşık 5000 mikron 2 adet 100 bölgelerden başlayarak, verim RNA 1.5 mikrogram oldu. Rastgele hekzamer ile cDNA sentez Super Senaryo III (Invitrogen) kullanarak, manuel diseksiyon Doğruluk sonra RT-PCR ile susturuldu ve unsilenced örnekleri GFP ifade kontrol tarafından kontrol edildi. Master Mix (Invitrogen) kullanarak Gerçek zamanlı RT-PCR ile düzenleyici yolun varsayılan hedefleri ile birlikte, susturuldu gen X ifade düzeylerini belirlemek için odaklanmış sonraki kantitatif analizleri yapıldı.

<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>

Unsilenced kanat disklerin ön / arka bölümlerinde elde bazal ekspresyon düzeyleri ile ilgili olarak, seçilen gen X faaliyet susturulması üzerine% 40 düşürüldü bulundu. Bunun aksine, olası hedefler (genler Y) biri bizi olumsuz X geni tarafından düzenlenir olduğu hipotezi doğrulamak için önde gelen (7 kat artış) up-regüle önemli ölçüde bulundu Biz, yukarıda açıklanan deneysel yaklaşım, çeşitli yasal yollar varsayılan hedeflerinin doğrulama başarıyla uygulan...

Suşları Fly Drosophila UAS-susturma hatları VDRC RNAi toplama 2 elde edilebilir. Bloomington Drosophila Menkul Kıymetler Merkezi (Indiana, ABD), GAL4 sürücü hatları bir koleksiyon mevcuttur. Ekipman Gerekli ekipman Lazer microdissector (Leica LMD6000), Coulter mikrofuge&#39;de 22R (Beckman), PCR (Benim Cycler BIO-RAD), Real Time PCR (BIO-RAD iQ5) içerir. Araçları Gerekli araçlar şunlardır: cam kuyular, fırça, mikrodiseksiyon forseps, asılı damla slaytlar, şırınga iğneleri, PET membran, plastik borular (50 ml, 05 ml, 0.25 ml) ile metal çerçeveler üzerine monte böcek pimleri.

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

Dokuların heterojen doğası, mansap analizleri ödün, biyolojik örnekler üretilen bilgi miktarı kısıtlayıcı bir faktör olduğu kanıtlanmıştır. In vivo etkileşimleri kapsamlı moleküler analiz özetlemek için, birçok dokuda içinde varolan karmaşık ve dinamik hücresel dernekler göz önüne alındığında biri kendi ana kapsamında özel hücre popülasyonlarının analiz etmek gerekir. Lazer-aracılı mikrodiseksiyon moleküler bütünlüğünü korurken ilgi hücreleri doğrudan mikroskobik görüntü altında açık kimliği ve başarılı bir hasat sağlayarak, bu hedefe ulaşabilirsiniz. Biz, büyüme kontrolü, hücre farklılaşması ve organogenez çalışma için avantajlı bir model sistemi temsil eden gelişmekte olan Drosophila kanat disk, tanımlı alanlar içinde gen ekspresyonu analiz etmek için bu teknolojiyi uyguladık. Larvaların hayali diskler precociously soy kısıtlama sınırları anterior ve posterior, dorsal ve ventral bölmelere ayrılmıştır. Indüklenebilir GAL4 UAS ikili ifade sistemi, bu bölmelerin her biri özel olarak inşa GAL4 uygun sürücünün kontrolü altında bir UAS-GFP transgen ifade transgenik sinekler etiketli olabilir kullanılması. Transgenik diskler, ayrık altkümelerini hücreleri gen ekspresyon profili tam olarak, lazer kesim kılavuzu floresan GFP sinyali kullanılarak, lazer aracılı mikrodiseksiyon sonra tespit edilebilir. Çeşitli aşağı uygulamaları arasında, lokalize RNA enterferans (RNAi) sonra RNA transkript profil üzerinde duruldu. GFP etiketleme, RNAi teknolojisi gelişiyle birlikte, belirli bir gen susturulması için ayrı bir hücre popülasyonu transkripsiyonel yanıt tespitine izin verilen bir genin lokalize demonte ile birleştiğinde olabilir. Bu yaklaşımı doğrulamak için, diskin posterior (GFP ifade etiketli) eşdeğer alanlar diseke ve bir başka yerde eksprese gen P bölmesi bölgesel susturulması üzerine anterior (etiketsiz) bölmesi. RNA microdissected susturulması ve unsilenced alanları ve kantitatif gerçek zamanlı RT-PCR tarafından belirlenen karşılaştırmalı gen ekspresyon profili çıkarılmıştır. Biz bu yöntem etkili Drosophila hayali diskler içinde hücreler altkümelerini doğru transkriptomik için uygulanabilir olduğunu göstermektedir. RNA kaynağı olarak büyük disk hazırlığı genellikle hücre homojenlik varsayar Nitekim, iyi transkripsiyonel ifade pozisyon bilgisi sonucunda bu yapıların içinde büyük ölçüde değişebilir bilinir. Lazer kesim kılavuzu lokalize floresan GFP sinyalini kullanarak, daha doğru transkripsiyonel analizleri yapılır ve kârlı onların yerli kapsamında farklı hücre soylarının transkript profil de dahil olmak üzere farklı uygulamaları, uygulanabilir.

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

Lazer mikrodiseksiyon Drosophila kanat diskin belirli bölümlerinde lokalize RNAi tabi gen ekspresyonu analiz etmek için uygulanan<em&gt; In vivo</em&gt;. RNA susturulması ve unsilenced bölmeleri eşdeğer alanlarında çıkarılan doğal dokuya microecology bağlamında karşılaştırmalı gen ekspresyon profili belirlemek için kantitatif RT-PCR ile analiz edildi.

Lazer Diseksiyon hücreleri Altküme Kaynak gösterilmeden Uygulanan Drosophila Kanat Disk

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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Biology

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

11.1K Görüntüleme.  University of Naples. Lazer mikrodiseksiyon Drosophila kanat diskin belirli bölümlerinde lokalize RNAi tabi gen ekspresyonu analiz etmek için uygulanan In vivo. RNA susturulması ve unsilenced bölmeleri eşdeğer alanlarında çıkarılan doğal dokuya microecology bağlamında karşılaştırmalı gen ekspresyon profili belirlemek için kantitatif RT-PCR ile analiz edildi.

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.

Memeli Doku Lazer Yakalama Mikrodisseksiyon

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

Biyoloji

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.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

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.

Parafin ve Dondurulmuş Bölüm Drosophila Yetişkin Kaslar

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.

Active T. in vitro Sulandırma castaneum Telomeraz

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.

Dan Tagged Çekirdeğin Affinity-tabanlı yalıtım<em&gt; Drosophila</emGen ekspresyon analizi için&gt; Dokular

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.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

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.

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

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.