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	<li>Griffiths-Jones, S., Grocock, R. J., van Dongen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+microRNA+sequences,+targets+and+gene+nomenclature.">miRBase: microRNA sequences, targets and gene nomenclature.</a> Nucleic Acids Res. 34, D140-D144 (2006).</li><li>Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+cancer+connection:+the+beginning+of+a+new+tale.">MicroRNA cancer connection: the beginning of a new tale.</a> Cancer Res. 66, 7390-7394 (2006).</li><li>Chen, J. F., Murchison, E. P., Tang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+Dicer+in+the+heart+leads+to+dilated+cardiomyopathy+and+heart+failure.">Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6-6 (2008).</li><li>Baek, D., Villen, J., Shin, 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+impact+of+microRNAs+on+protein+output.">The impact of microRNAs on protein output.</a> Nature. 455, 64-71 (2008).</li><li>Hendrickson, D. G., Hogan, D. J., Herschlag, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+mRNAs+recruited+to+argonaute+2+by+specific+microRNAs+and+corresponding+changes+in+transcript+abundance.">Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.</a> PLoS One. 3, e2126-e2126 (2008).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Lall, S., Krek, A., Chen, K. <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+map+of+conserved+microRNA+targets+in.">A genome-wide map of conserved microRNA targets in.</a> 16, 460-471 (2006).</li><li>Boutz, D. R., Collins, P. J., Suresh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-tiered+approach+identifies+a+network+of+cancer+and+liver+disease-related+genes+regulated+by+miR-122.">Two-tiered approach identifies a network of cancer and liver disease-related genes regulated by miR-122.</a> J. Biol. Chem. 286, 18066-18078 (2011).</li><li>Lim, L. P., Lau, N. C., Garrett-Engele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+shows+that+some+microRNAs+downregulate+large+numbers+of+target+mRNAs.">Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.</a> Nature. 433, 769-773 (2005).</li><li>Selbach, M., Schwanhausser, B., Thierfeldner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+changes+in+protein+synthesis+induced+by+microRNAs.">Widespread changes in protein synthesis induced by microRNAs.</a> Nature. 455, 58-63 (2008).</li><li>Hutvagner, G., Simard, M. J., Mello, C. C., Zamore, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+inhibition+of+small+RNA+function.">Sequence-specific inhibition of small RNA function.</a> PLoS Biol. 2, e98-e98 (2004).</li><li>Meister, G., Landthaler, M., Dorsett, Y., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+inhibition+of+microRNA-+and+siRNA-induced+RNA+silencing.">Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing.</a> RNA. 10, 544-5450 (2004).</li><li>Liao, J. M., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auto-regulatory+suppression+of+c-Myc+by+miR-185-3p.">Auto-regulatory suppression of c-Myc by miR-185-3p.</a> J. Biol. Chem. Epub. , (2011).</li></ol>

الكتاب والعاملين في جينوم مفاتيح الذين يصنعون الأدوات والكواشف التي تظهر في هذه المقالة. وقد رعت الإنتاج من هذه المادة بواسطة مفاتيح علم الجينوم.

وقد انشأنا مجموعة واسعة من الخريطة الوراثية البشرية المستنسخة قبل يبني 3&#39;UTR - مراسل البشرية لتمكينها من الدراسات ميرنا - 3&#39;UTR التفاعلات في الخلايا الحية. هنا أظهرنا طريقة استنساخه وعالية الإنتاجية من أجل يحاكي ميرنا التعاون مع transfecting 3&#39;UTR - صحفيين باستخدام نظام مراسل luciferase. هذا الأسلوب يعتمد على الباحثين باستخدام خط خلية عالية transfectable والاقلاق النشاط ميرنا مع تقنية overexpression. يمكن للباحثين باستخدام خطوط الخلية سيئة transfectable النظر في استخدام لدينا رفيق lentiviral 3&#39;UTR - مراسل جمع إنشاؤها بالتعاون مع سيغما (بعثة 3&#39;UTR Lenti GoClones). علاج الخلايا مع ميرنا يحاكي النتائج في تجربة overexpression من نوع ، وبأي دراسة من هذا القبيل ، والباحثين يجب أن تأخذ بعين الاعتبار أن هذه قد تكون أو لا تمثل الظروف الفسيولوجية الفعلية. لمعالجة الشواغل overexpression ، يمكن للباحثين استخدام موانع ميرنا بالإضافة إلى يحاكي لدراسة التفاعل مع اثنين من أنواع مختلفة من اضطراب 12-14. حالما يتم تحديد الأهداف ميرنا الأرجح ، قد ترغب في mutagenize الباحثين المواقع المفترضة ميرنا ملزمة لتحديد ما إذا كانت المواقع اللازمة للتفاعل وظيفي. قد يرغبون أيضا في رفع مستوى لاختبار المئات من مختلف UTRs هدفا محتملا (Boutz). بالإضافة إلى ذلك ، لو قيض للباحثين والمهتمين حول ما إذا كان يؤثر على الاستقرار ميرنا نص الترجمة أو كفاءة ، يمكن مقارنة البيانات مراسل luciferase لتدابير مستويات نص ثابت للدولة. فإن أي تغيير في إشارة luciferase التي لا ترتبط مع تغيير نسبي في مستويات النص إشارة إلى التأثير على كفاءة الترجمة. أخيرا ، يمكن للباحثين استخدام ناقلات مراسل إنشاء منحنى الاستجابة للجرعة مقارنة الحساسية النسبية لمختلف UTRs للتغيرات في تركيز ميرنا 14.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> بائع </td><td> رقم كتالوج </td></tr><tr><td> لوحات بيضاء زراعة الأنسجة (96 - جيدا أسفل الصلبة) </td><td> VWR </td><td> 82050-736 </td></tr><tr><td> لوحات واضحة زراعة الأنسجة (96 أيضا) مع غطاء </td><td> VWR </td><td> 353072 </td></tr><tr><td> LightSwitch الكاشف الفحص Luciferase </td><td> مفاتيح </td><td> LS010 </td></tr><tr><td> DharmaFECT الكاشف ترنسفكأيشن ديو (0.75mL) </td><td> Dharmacon </td><td> T - 2010-02 </td></tr><tr><td> احباط ختم الشريط بلايت </td><td> E &amp; K العلمية </td><td> T592100 </td></tr><tr><td> تنفس بلايت ختم الشريط </td><td> E &amp; K العلمية </td><td> T896100 - S </td></tr><tr><td> لوحة Luminometer </td><td> الأجهزة الجزيئية </td><td> SpectraMax L </td></tr></tbody></table>

identifying targets of human micrornas with the lightswitch luciferase assay system using 3'utr-reporter constructs and a microrna mimic in adherent cells

MicroRNAs (miRNAs) هي مهمة للتعبير المنظمين والجينات تلعب دورا في العديد من العمليات البيولوجية. وقد تم تحديد أكثر من 700 miRNAs الإنسان حتى الآن مع كل وجود ما يصل الى مئات mRNAs الهدف فريدة من نوعها. الأدوات الحسابية ، المقايسات التعبير والبروتيوميات وتقنيات الكروماتين مناعي القائم على توفير ادلة هامة لتحديد mRNAs التي أهدافا مباشرة لميرنا معينة. بالإضافة إلى ذلك ، أصبحت 3&#39;UTR مراسل المقايسات ، عنصرا هاما من عناصر دراسات مستفيضة ميرنا الهدف لأنها توفر أدلة وظيفية للquantitate آثار التفاعلات ميرنا - 3&#39;UTR محددة في النظام القائم على الخلية. لتمكين المزيد من الباحثين على المقايسات 3&#39;UTR مراسل النفوذ ، ودعم النطاق تصل إلى مثل هذه المقايسات والإنتاجية العالية المستوى ، وقد انشأنا مجموعة واسعة من الجينوم البشري للصحفيين 3&#39;UTR luciferase في Luciferase LightSwitch عالي الكفاءة فحص النظام. ويتضمن النظام أيضا بنيات تركيبية مراسل ميرنا هدف لاستخدامها الضوابط الإيجابية ، ومختلف مراسل 3&#39;UTR يبني الذاتية ، وسلسلة من البروتوكولات التجريبية موحدة. نحن هنا تصف طريقة لترنسفكأيشن المشترك للبنيات 3&#39;UTR - المراسل الفرد جنبا الى جنب مع ميرنا تقليد تتسم بالكفاءة واستنساخه ، وقابلة للتحليل إنتاجية عالية.

Watch this Scientific Journal Video about Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells at 

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) هي منظمات هامة في التعبير الجيني ، وثبت أنها تلعب دورا في العديد من العمليات البيولوجية. إلى فهم أفضل لميرنا ، UTR التفاعلات ، وقد انشأنا مجموعة واسعة من الجينوم للصحفيين UTR 3 luciferase يقترن جينة luciferase الرواية وكاشف مقايسة ، ونظام LightSwitch.

تحديد الأهداف من microRNAs الإنسان مع نظام الفحص LightSwitch Luciferase به مراسل 3&#39;UTR - a يبني وmicroRNA مقلد في خلايا منتسب

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

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Biology

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

36.0K Views.  SwitchGear Genomics. MicroRNAs (miRNAs) هي منظمات هامة في التعبير الجيني ، وثبت أنها تلعب دورا في العديد من العمليات البيولوجية. إلى فهم أفضل لميرنا ، UTR التفاعلات ، وقد انشأنا مجموعة واسعة من الجينوم للصحفيين UTR 3 luciferase يقترن جينة luciferase الرواية وكاشف مقايسة ، ونظام LightSwitch.

Video: Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

دراسة النشوء الأحيائي الأغشية مع الفحص الجيني مراسل Luciferase المستندة

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

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.

باستخدام الجينات Electroporation MXcell بولسير النظام لTransfect الخلايا الأولية ذات الكفاءة العالية

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

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

في تقطير التحت خلوية الموقع من خلايا الثدييات وغير ملتصقة منتسب -

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

كفاءة اشتقاق السلائف القلب البشري وافرة القوة العضلية من خلايا المنشأ الجنينية البشرية التعريفي مع جزيء صغير

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

إعادة برمجة الخلايا الجسدية البشرية المستحثة إلى الخلايا الجذعية المحفزة (iPSCs) استخدام ناقلات فيروسات الرجعية مع GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

مقايسة الوظيفية للفجوة صلي فحص. Electroporation للمن الخلايا الملتصقة على الإنديوم أكسيد القصدير

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

الكشف عن أهداف ميرنا في إنتاجية عالية باستخدام الفحص 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.