משכורות לתמוך במחקר סופקו על ידי מועצת סויה הברית, על ידי קרנות המדינה הפדרלי השתלט על המחקר באוניברסיטת אוהיו חקלאי / אוהיו מרכז פיתוח. מחקר זה גם נתמך בחלקו על ידי מענק מן CONACYT, מקסיקו, כדי CMHG. ציון של סימן מסחרי או מוצרים קנייניים אינו מהווה ערובה או אחריות של המוצר על ידי OSU / OARDC וגם אינה מעידה על אישור למעט מוצרים אחרים כי יכול להיות גם מתאים. כתב העת מאמר לא HCS 09-17.

<ol>
	<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol Plant. 15, 473-497 (1962).</li><li>Gamborg, O. L., Miller, R. A., Ojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+requirements+of+suspension+cultures+of+soybean+root+cells.">Nutrient requirements of suspension cultures of soybean root cells.</a> Exp Cell Res. 50, 150-158 (1968).</li><li>Finer, J. J., Vain, P., Jones, M. W., McMullen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+particle+inflow+gun+for+DNA+delivery+to+plant+cells.">Development of the particle inflow gun for DNA delivery to plant cells.</a> Plant Cell Rep. 11, 232-238 (1992).</li><li>Finer, J. E., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+reducing+moisture+condensation+on+Petri+dish+lids.">A simple method for reducing moisture condensation on Petri dish lids.</a> Plant Cell Tiss Org. 91, 299-304 (2007).</li><li>Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+35S+and+Lectin+promoters+in+transgenic+soybean+tissue+using+an+automated+image+acquisition+system+and+image+analysis.">Comparative analysis of 35S and Lectin promoters in transgenic soybean tissue using an automated image acquisition system and image analysis.</a> Plant Cell Rep. 25, 290-296 (2006).</li><li>Chiera, J. M., Bouchard, R. A., Dorsey, S. L., Park, E. H., Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+two+highly+active+soybean+(Glycine+max+(L.)+Merr.)+promoters+and+their+characterization+using+a+new+automated+image+collection+and+analysis+system.">Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system.</a> Plant Cell Rep. 26, 1501-1509 (2007).</li><li>Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+extension+of+transient+GFP+expression+by+the+co-introduction+of+a+suppressor+of+silencing.">Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing.</a> Transgenic Res. 17, 1143-1154 (2008).</li><li>Dhillon, T., Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+six+different+viral+suppressors+of+silencing+using+image+analysis+of+transient+GFP+expression.">Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression.</a> Plant Cell Rep. 28, 639-647 (2009).</li></ol>

השימוש ברובוטיקה יש יישומים עצום היבטים שונים של חיי האדם, במיוחד, רובוטים שימשו ביעילות לבצע פעולות בסביבה מסוכנת, כדי להפוך פעילות מייגע ומורכב, וכדי לבצע משימות באופן מדויק יותר. בביולוגיה מולקולרית, ובמיוחד ביטוי מנתח גן, רובוטים יכולים לעזור לעקוב לא רק תכונות של גנים, אלא גם לצמיחה של רקמה ופיתוח לאורך זמן. תופעות ביולוגיות רבות מתרחשות באופן דינמי, אשר עשוי להיות קשה לעקוב באמצעות נקודה אחת התצפיות זמן. השימוש של הגן GFP ללימודי הביטוי מביא יתרונות נוספים עבור צפייה בתגובה רקמות וצמיחה. עבור פרוצדורות הניסוי שלנו, GFP מאפשרת לנו לעקוב אחר ביטוי גנים אותה פיסת רקמה לאורך זמן כמו גילוי ה-GFP הוא הרסני. יתר על כן, הגרסה שלנו של חלבון ה-GFP היא יציבה מספיק כדי לאפשר זיהוי, אלא גם מראה כמה מחזור כדי למזער את הצטברות ברקמות צמח, המאפשר לנו לעקוב גם את עלייתה ונפילתה של ביטוי גנים. יש לנו כבר אוסף מנוצל תמונה רובוטית שלנו מערכת ניתוח עבור מגוון רחב של יישומים. אנו צופים פוטנציאל גבוה עבור יישומים ביולוגיים רבים בהם הבנה דינמית של תופעה הוא הרצוי. לדוגמה, צמיחה והתפתחות של רקמות הצמח ניתן לעקוב באמצעות המערכת שלנו נותנת תובנה חשובה / מידע על תהליכים אלה. כמו כן, הדינמיקה של חלבון הובלה באמצעות הגנים הכתב כמו ה-GFP, ניתן דמיינו בקלות באמצעות זמן לשגות אנימציות. המתודולוגיה שתוארו בדו&quot;ח זה הוא מורכב טכנית, אבל פשוטה מבחינה מושגית. התוצאות שלנו הם חזקים ויישומים חדשים ממשיכים להתגלות.

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<td>ImageJ</td>
<td>Software</td>
<td>U. S. National Institutes of Health</td>
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<td><a href="http://www.rsb.info.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></td>
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robotics and dynamic image analysis for studies of gene expression in plant tissues

התבטאות גנים ברקמות הצמח הוא למד בדרך כלל על ידי מיצוי ההרסנית של תרכובות מרקמות הצמח<em&gt; במבחנה</em&gt; מנתח. השיטות שהוצגו כאן לנצל את חלבון פלואורסצנטי ירוק (<em&gt; GFP</em&gt;) גן לניטור מתמיד של ביטוי גנים החלקים זהה של רקמות, לאורך זמן.<em&gt; GFP</emגן&gt; הושם תחת שליטה הרגולציה של יזמים שונים שהוכנסו שעועית לימה רקמות cotyledonary באמצעות הפצצה החלקיקים. Cotyledons הונחו לאחר מכן על מערכת רובוטית אוסף תמונות, שכללה מיקרוסקופ פלואורסצנטי לנתח עם מצלמה דיגיטלית פלטפורמה 2 ממדית ורובוטיקה אישית מעוצבת על מנת לאפשר התקשרות בטוחה של מנות התרבות. תמונות שנאספו רקמות cotyledonary כל שעה במשך 100 שעות כדי לייצר פרופילי ביטוי עבור מקדם כל אחד. כל סדרה של אסף 100 תמונות היה נתון הראשונה יישור התמונה ידנית באמצעות ImageReady לוודא GFP-לבטא מוקדים היו שמר בעקביות בתוך השדות שנבחרו לניתוח. אזורים ספציפיים של הסדרה מדידה 300 x 400 פיקסלים, אז נבחרו לניתוח נוסף כדי לספק מדידות אינטנסיביות GFP באמצעות תוכנת ImageJ. תמונות אצווה הופרדו הערוצים אדום, ירוק וכחול GFP-לבטא אזורים זוהו באמצעות התכונה סף ImageJ. לאחר הפחתת הקרינה רקע (חיסור של ערכי האפור של אי - הבעת פיקסלים של כל פיקסל) בערוצים אדום וירוק בהתאמה, עוצמת ה-GFP היה מחושב על ידי הכפלת ממוצע בגווני אפור ערך לפיקסל במספר הכולל של ה-GFP-לבטא פיקסלים בכל הערוץ, ולאחר מכן להוסיף אותם ערכים עבור שני הערוצים אדום וירוק. ערכי ה-GFP אינטנסיביות נאספו על כל 100 נקודות זמן תשואה פרופילי הביטוי. וריאציות פרופילים GFP הביטוי נובע מהבדלים גורמים כגון חוזק האמרגן, נוכחות של מדכאי השתקה או לאופי של האמרגן. בנוסף כימות של עוצמת ה-GFP, הסדרה תמונה שימשו גם ליצירת זמן לשגות אנימציות באמצעות ImageReady. זמן לשגות אנימציות גילה כי רוב ברור של תאים מוצגת עלייה מהירה יחסית ביטוי של GFP, ואחריו ירידה איטית. תאים מסוימים המוצגים לעתים אובדן פתאומי של הקרינה, אשר עשוי להיות קשור עם מוות של תאים מהירה. תחבורה לכאורה של ה-GFP על פני קרום התא ועל הקיר לתאים סמוכים נצפתה גם. זמן לשגות אנימציות מידע נוסף ובלבד לא יכול להיות אחרת שהושגו באמצעות עוצמת GFP פרופילים או יחידה נקודת זמן אוספים התמונה.

Watch this Scientific Journal Video about Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues at JoVE.com

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

אנו מדווחים על שיטה הקדמה, מעקב וניתוח כמותי של ה-GFP ביטוי בתאי הצמח. שיטה זו משתמשת מערכת אישית מעוצבת רובוטיקה לאיסוף חצי תמונה רציפה של מספר גדול של דגימות, לאורך זמן. אנחנו גם מדגימים את השימוש ImageJ ו ImageReady ניתוח של סדרת תמונות.

רובוטיקה ניתוח תמונה דינאמית ללימודים של ביטוי גנים ברקמות הצמח

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

robotics-dynamic-image-analysis-for-studies-gene-expression-plant

Nanyang Technological University

Research

JoVE Journal

Biology

12.5K Views.  The Ohio State University. אנו מדווחים על שיטה הקדמה, מעקב וניתוח כמותי של ה-GFP ביטוי בתאי הצמח. שיטה זו משתמשת מערכת אישית מעוצבת רובוטיקה לאיסוף חצי תמונה רציפה של מספר גדול של דגימות, לאורך זמן. אנחנו גם מדגימים את השימוש ImageJ ו ImageReady ניתוח של סדרת תמונות.

Video: Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

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.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור 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 ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virus-Induced להשתקת גנים (VIGS) ב ניקוטיאנה benthamiana עגבניות

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

חוט השדרה Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

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.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים 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.

מדורים פרפין-Embedded קפואים של תסיסנית סקס שרירים

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.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

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.

בידוד מבוסס זיקה של תייג גרעינים מ<em&gt; דרוזופילה</em&gt; רקמות לניתוח ביטוי גנים

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.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

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

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

ניתוח האוטומטי של דינמי Ca<sup&gt; 2 +</sup&gt; אותות ברצפי תמונות

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...