Ce travail a été soutenu par des subventions de la Fondation de Pittsburgh et le National Institutes of Health (NIH) [GM087798; CA148629; ES019498] pour RWS. Prise en charge de l&#39;UPCI Facilité Lentiviral a été fournie à RWS par la subvention d&#39;appui du Centre du cancer de la National Institutes of Health [P30 CA047904]. Soutien a également été fourni par le ministère Université de Pittsburgh de pharmacologie et de biologie chimique à la DS. Soutien a également été fourni par l&#39;ESTRO à CV.

<ol>
	<li>Mutamba, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XRCC1+and+base+excision+repair+balance+in+response+to+nitric+oxide.">XRCC1 and base excision repair balance in response to nitric oxide.</a> DNA Repair (Amst). 10, 1282-1293 (2011).</li><li>Tang, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methylpurine+DNA+glycosylase+and+DNA+polymerase+beta+modulate+BER+inhibitor+potentiation+of+glioma+cells+to+temozolomide.">N-methylpurine DNA glycosylase and DNA polymerase beta modulate BER inhibitor potentiation of glioma cells to temozolomide.</a> Neuro-oncology. 13, 471-486 (2011).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+and+nucleic+acids.">Fluorescence resonance energy transfer and nucleic acids.</a> Methods Enzymol. 211, 353-388 (1992).</li><li>Yaron, A., Carmel, A., Katchalski-Katzir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecularly+quenched+fluorogenic+substrates+for+hydrolytic+enzymes.">Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes.</a> Anal. Biochem. 95, 228-235 (1979).</li><li>Kreklau, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorometric+oligonucleotide+assay+to+measure+O(+6)-methylguanine+DNA+methyltransferase,+methylpurine+DNA+glycosylase,+8-oxoguanine+DNA+glycosylase+and+abasic+endonuclease+activities:+DNA+repair+status+in+human+breast+carcinoma+cells+overexpressing+methylpurine+DNA+glycosylase.">A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase.</a> Nucleic Acids Res. 29, 2558-2566 (2001).</li><li>Belon, C. A., Frick, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+helicase+activity+with+molecular+beacons.">Monitoring helicase activity with molecular beacons.</a> BioTechniques. 45, 433-442 (2008).</li><li>Ma, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+DNA+polymerase+activity+using+molecular+beacon.">Real-time monitoring of DNA polymerase activity using molecular beacon.</a> Anal. Biochem. 353, 141-143 (2006).</li><li>Summerer, D., Marx, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+for+quantitative+monitoring+of+the+DNA+polymerase+reaction+in+real-time.">A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time.</a> Angew. Chem. Int. Ed. Engl. 41, 3620-3622 (2002).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+beacon+to+monitor+activity+of+E.+coli+DNA+ligase.">Using molecular beacon to monitor activity of E. coli DNA ligase.</a> The Analyst. 130, 350-357 (2005).</li><li>Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+beacons+for+the+detection+of+DNA+and+telomerase+activity.">Catalytic beacons for the detection of DNA and telomerase activity.</a> J. Am. Chem. Soc. 126, 7430-7431 (2004).</li><li>Kundu, L. M., Burgdorf, L. T., Kleiner, O., Batschauer, A., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavable+substrate+containing+molecular+beacons+for+the+quantification+of+DNA-photolyase+activity.">Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.</a> Chembiochem : a European journal of chemical biology. 3, 1053-1060 (2002).</li><li>Sokol, D. L., Zhang, X., Lu, P., Gewirtz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+detection+of+DNA.RNA+hybridization+in+living+cells.">Real time detection of DNA.RNA hybridization in living cells.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543 (1998).</li><li>Yang, X., Tong, C., Long, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+fluorescence+assay+for+hSMUG1+activity+based+on+modified+molecular+beacon.">A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon.</a> Molecular and cellular probes. 25, 219-221 (2011).</li><li>Mirbahai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+molecular+beacon+to+track+the+activity+of+base+excision+repair+protein+OGG1+in+live+cells.">Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.</a> DNA Repair (Amst). , (2009).</li><li>Maksimenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+assay+for+measuring+base+excision+repair+activities.">A molecular beacon assay for measuring base excision repair activities.</a> Biochem. Biophys. Res. Commun. 319, 240-246 (2004).</li></ol>

RWS est un consultant scientifique pour Trevigen, Inc Le reste des auteurs déclarent qu&#39;il n&#39;ya pas de conflits d&#39;intérêts.

Il ya plus de 600 citations à l&#39;aide «molecular beacon» le terme pour la détection et la quantification de nombreuses molécules et les activités enzymatiques, y compris l&#39;activité hélicase 6, une activité ADN polymérase 7,8, l&#39;ADN ligase l&#39;activité 9, 10 activité de la télomérase, l&#39;ADN photolyase d&#39;activité de 11 et de l&#39;ADN / ARN hybrides 12, parmi beaucoup d&#39;autres. En plus de l&#39;utilisation de balises moléculair...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Chlorure de potassium </td><td> Pêcheur </td><td> P217-500 </td><td> Pour la biologie moléculaire </td></tr><tr><td> EDTA </td><td> Pêcheur </td><td> BP120-500 </td><td></td></tr><tr><td> Glycérol </td><td> Pêcheur </td><td> BP229-1 </td><td> Pour la biologie moléculaire </td></tr><tr><td> TNT </td><td> Pêcheur </td><td> BP172-5 </td><td></td></tr><tr><td> HEPES-Solution </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> HEPES-Sel </td><td> Sigma </td><td> H4034-500G </td><td></td></tr><tr><td> Hydroxyde de potassium </td><td> Sigma </td><td> 17-8 </td><td></td></tr><tr><td> 0.22μM filtre </td&gt; <td> Nalgene </td><td> 167-0020 </td><td></td></tr><tr><td> Produits de dialyse Slide-A-Lyzer </td><td> Percer </td><td> 66373 </td><td> MW 7000 </td></tr><tr><td> Seringue </td><td> Pêcheur </td><td> 309659 </td><td></td></tr><tr><td> Aiguille </td><td> Pêcheur </td><td> 305196 </td><td></td></tr><tr><td> 1,5 ml Microtubes à centrifuger </td><td> Pêcheur </td><td> 05-408-129 </td><td> Noir </td></tr><tr><td> 15 ml Falcon Tubes </td><td> Pêcheur </td><td> 352097 </td><td></td></tr><tr><td> Kit d&#39;extraction de protéines NucBuster </td><td> Calbiochem </td><td> 71183 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 14190 </td><td></td></tr><tr><td> Beacons moléculaires </td><td> IDT </td><td></td><td></td></tr><tr><td> BioRad Protein dosage colorant reConcentré d&#39;agent </td><td> BioRad </td><td> Cat # 500-0006 </td><td></td></tr></tbody></table>

quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons

Nous décrivons une méthode pour la détermination quantitative, mesure en temps réel de l&#39;ADN glycosylase et des activités endonucléase AP dans des lysats de cellules nucléaires de base à l&#39;aide de réparation excision (BER) des balises moléculaires. Le substrat (balise) est constitué d&#39;un désoxyoligonucléotide contenant une lésion de base unique avec une 6-carboxyfluorescéine (6-FAM) fragment conjugué à l&#39;extrémité 5 &#39;et un fragment Dabcyl conjugué à l&#39;extrémité 3&#39; de l&#39;oligonucléotide. La balise BER moléculaire est de 43 bases de longueur et la séquence est conçu pour favoriser la formation d&#39;une structure tige-boucle avec 13 nucléotides dans la boucle et 15 paires de bases dans la tige 1,2. Lorsqu&#39;elle est pliée dans cette configuration, le groupe 6-FAM est désactivé par Dabcyl de manière non fluorescent par l&#39;intermédiaire d&#39;Förster résonance de l&#39;énergie de transfert (FRET) 3,4. La lésion est positionné de telle sorte que après le retrait lésion de base et la scission brin restants 5 oligonucléotide de base contenant le groupe 6-FAM est libéré de la tige. Libérer undétachement e des quencher (DABCYL) entraîne une augmentation de la fluorescence qui est proportionnelle au niveau de la réparation de l&#39;ADN. En recueillant de multiples lectures des valeurs de fluorescence, en temps réel d&#39;évaluation de l&#39;activité BER est possible. L&#39;utilisation de la norme quantitative instruments PCR en temps réel permet l&#39;analyse simultanée de nombreux échantillons. La conception de ces balises BER moléculaires, avec une lésion de base unique, est soumise à des analyses cinétiques, la quantification et la validation BER inhibiteur et est adaptable pour la quantification de l&#39;activité réparation de l&#39;ADN dans les tissus et les lysats de cellules tumorales ou avec des protéines purifiées. L&#39;analyse de l&#39;activité BER dans les lysats tumoraux ou de tissus aspire à l&#39;aide de ces balises moléculaires peuvent être applicables à des mesures de biomarqueurs fonctionnels. En outre, l&#39;analyse de l&#39;activité du TEB avec des protéines purifiées en utilisant ce dosage quantitatif fournit un moyen rapide, à haut débit méthode pour la découverte et la validation des inhibiteurs de BER.

Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Nous décrivons une méthode pour la détermination quantitative, mesure en temps réel de l&#39;ADN glycosylase et des activités endonucléase AP dans des lysats de cellules nucléaires. Le test donne des taux d&#39;activité de réparation d&#39;ADN qui se prêtent à l&#39;analyse cinétique et est adaptable pour la quantification de l&#39;activité réparation de l&#39;ADN dans les tissus et les lysats tumoraux ou avec des protéines purifiées.

Quantitative, en temps réel Analyse de l&#39;activité de réparation par excision de base dans les lysats cellulaires Utilisant des lésions spécifiques Beacons moléculaires

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

quantitative-real-time-analysis-base-excision-repair-activity-cell

Research

JoVE Journal

Biology

13.5K vues.  University of Pittsburgh School of Medicine. Nous décrivons une méthode pour la détermination quantitative, mesure en temps réel de l'ADN glycosylase et des activités endonucléase AP dans des lysats de cellules nucléaires. Le test donne des taux d'activité de réparation d'ADN qui se prêtent à l'analyse cinétique et est adaptable pour la quantification de l'activité réparation de l'ADN dans les tissus et les lysats tumoraux ou avec des protéines purifiées.

Vidéo: Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

Quantitative PCR en temps réel utilisant le Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Recherche

Biologie

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Analyse de l&#39;expression génique dans l&#39;agrile du frêne ( Agrilus planipennis) En utilisant quantitative en temps réel-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Profilage de la pré-micro ARNs et micro-ARN en utilisant quantitative PCR en temps réel (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Application de la MassSQUIRM pour des mesures quantitatives de l&#39;activité Déméthylase Lysine

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Un flux de travail de remise en forme Analyse quantitative

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Analyse en temps réel de profil métabolique dans<em&gt; Ex Vivo</em&gt; Cultures souris intestinale Crypte organoïde

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

PCR en temps réel basée sur une sonde Approches de mesure quantitative des microARN

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analyse qPCRTag - un débit élevé, PCR temps réel pour Sc2.0 Génotypage

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...