1. Die Thermo Scientific Solaris qPCR Assay-Design-Algorithmus <ol><li> Der Algorithmus in das Design der Solaris qPCR Sonde / Primerpaare verwendet passt die Tm jeder Komponente wodurch universelle Radfahren Bedingungen. Der Einbau der MGB, oder kleine Furche binden, Einheit in der Sonde erhöht die Tm und ermöglicht kürzere Sonden, um eine größere Assay Flexibilität und Leistung ausgelegt sein. Wie ihr Name schon sagt, sind MGBs eine Klasse von Antibiotika, die in die kleine Furche der DNA-Helix passt. MGBs sind die 3 oder 5 Ende einer DNA-Sonde befestigt. In Lösung wird die FAM-Reporter Fluoreszenz der Sonde von Eclipse Dunkle Quencher gequencht. Wenn die Sonde an eine Zielsequenz bindet, kann die daraus resultierende Veränderung in der Sonde s 3-D-Konformation ein starkes Fluoreszenzsignal. Die Bindung der DNA-Sonde zur Zielsequenz wird durch die MGB, die den Einsatz von hochspezifischen, kürzere Sonden ermöglicht und gleichzeitig die entsprechende Schmelztemperatur für PCR stabilisiert. So tritt Signalerkennung beim Glühen Phase der Target-Amplifikation. Während der Erweiterung Schritt distanziert die MGB-Sonde und die Fluoreszenz wieder abgeschreckt. Weil die Sonde distanziert, hat dies keine Auswirkungen PCR-Effizienz. </li><li> Ein weiteres Merkmal der Solaris-Algorithmus ist die Verwendung von Superbases. Superbases sind Grundlagen, die verbindliche Fähigkeiten verbessert haben, um die Stabilität von AT-Anleihen zu verbessern und zu beseitigen GG Selbstassoziation in Guanin reiche Sequenzen modifiziert. Hinzu kommt, dass sie nicht auslöschen benachbarten Fluorophoren an der 5-Terminus angehängt. Diese Basen sind selektiv platziert, nach dem Algorithmus, um die Verfügbarkeit von Zielsequenzen zu erweitern. Dies macht es möglich, Sequenzen, wie GC-reiche Regionen, die sonst in der Gestaltung Primer und Sonden vermieden werden verwenden würde. </li><li> Wann immer möglich, enthält die Solaris-Algorithmus Exon Junction Spanning Regionen in der Ziel-Site. Dies erhöht die Spezifität durch die Vermeidung der Verstärkung der Verunreinigung genomischer DNA. </li><li> Wichtig ist, dass der Algorithmus identifiziert auch eine Konsensus-Sequenz für alle bekannten Splice-Varianten, so dass alle von einer einzigen Sonde / Primer-Set abgedeckt sind. So ist nur ein Test für ein bestimmtes Gen von Interesse erforderlich. </li><li> Schließlich nutzt das Solaris qPCR Assay-Design-Algorithmus sehr strenge Parameter für kritische Überlegungen zum Entwurf einschließlich GC-Gehalt, Länge, Tm, und erstreckt sich von homogenen Nukleotiden. Die resultierenden Sequenzen BLAST für die Spezifität mit 3 verschiedenen Datenbanken analysiert: genomische, Transkript, und Pseudogen. </li></ol> 2. Beziehen Solaris Reagenzien <ol><li> Um die entsprechende Solaris Reagenzien, besuchen <a href="http://dharmacon.com">www.thermo.com / solaris</a> </li><li> Sobald Sie auf der Website auf &quot;GENEius Produktsuche&quot; klicken. Um direkt zu den GENEius Produkt zu suchen, gehen <a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com / SolarisSearch</a> </li><li> Klicken Sie in der Suchanfrage ein und wählen Sie den entsprechenden Zielorganismen. </li><li> Die GENEius Produktsuche verarbeiten kann viele verschiedene Gen-IDs. Geben Sie hier eine der vorgeschlagenen Suchbegriffen, um das Gen von Interesse, einschließlich Ref Seq Beitritt, der Gene-ID, die Gene Symbol, ein Gene Beschreibung der Sanger-ID oder Zugangsnummer oder eine Katalog-Nummer zu finden. Dann auf &quot;Suchen&quot; klicken. </li><li> Identifizieren des Gens von Interesse und klicken Sie auf &quot;Auswählen&quot; auf der linken Seite. Es öffnet sich ein Fenster, das die verschiedenen Thermo Scientific Produkte für das Gen von Interesse zeigt. Unter &quot;Produkte&quot; die Option &quot;qPCR Detection&quot; Tab. </li><li> Unter dem &quot;Solaris Gene Expression Assay&quot; Überschrift, sehen Sie eine optimal Solaris Sonde / der Primer-Assay entwickelt. Dieser Test deckt alle bekannten Spleißvarianten des Gens Ziel. Wählen Sie die gewünschte Menge. Sie sind in 100, 200, 400 zur Verfügung, und 1000 X 25 ul Reaktionen (wenn der Test als made-to-order identifiziert wird, die 100 Packungsgröße nicht verfügbar). Klicken Sie auf &quot;In den Warenkorb&quot;. </li><li> Als nächstes wählen Sie den entsprechenden Master-Mix für den Thermocycler, die verwendet werden. Um festzustellen, welche Master-Mix funktioniert am besten mit der Maschine zur Verfügung, klicken Sie auf &quot;Finden Sie heraus, welche Master Mix am besten mit Ihrem PCR Cycler!&quot; </li><li> Finden Sie die qPCR-Cycler, dass auf der Speisekarte verwendet werden, um festzustellen, welcher Mix zu verwenden. </li><li> Zurück zur GENEius Produktsuche Fenster, und wählen Sie den entsprechenden Master-Mix. Der Master-Mix kommt in 100, 200, 400 und 1000 Reaktion Größen. Klicken Sie auf &quot;In den Warenkorb&quot;. </li><li> Zitiervorschlag Gene und verso cDNA-Synthese-Kits können auch für eine vollständige qRT-PCR-Analyse bestellt werden. </li><li> Wenn das Solaris-qPCR-Reagenzien kommen, bewahren Sie sie bei -20 ° C bis zur Verwendung. Die folgenden Reagenzien empfangen werden soll: Ein 20fach-Lösung mit zwei Primern bei 800 nm und je einem Gen spezifischen Sonde bei 200 nm, sequ vorgesehenENCE Informationen sowohl für die Sonde und die Primer-und Solaris-qPCR Gene Expression Master Mix in einer 2X-Konzentration. Solaris qPCR Gene Expression Assays sind für mindestens 12 Monate stabil. Vermeiden Sie wiederholtes Einfrieren und Auftauen. </li></ol> 3. qPCR-Setup <ol><li> Beginnen Sie mit der qPCR-Protokoll mit cDNA. Gesamt-RNA extrahiert sollte mit einem Spin-Säule Methode. Die Thermo Scientific Verso cDNA-Synthese-Kit ist für die reverse Transkription in cDNA zu erzeugen empfohlen. </li><li> Bereiten Sie sich für qPCR durch Auftauen der Primer, Sonden und Master-Mix auf Eis. Auch zur Hand haben PCR-Wasser und Deckweiß qPCR-Platten, die die Sensitivität der Real-time PCR zu verbessern. </li><li> Der Master-Mix enthält alle Komponenten für die Echtzeit-PCR einschließlich: 1) Thermo-Start-DNA-Polymerase, ein Hot-Start-Version von Thermoprime DNA-Polymerase. Diese Art der Polymerase wird verwendet, um unspezifische Amplifikation während der Reaktion-Einrichtung zu vermeiden und erfordert die Aktivierung bei 95 ° C 15 min. 2) dNTPs in der Solaris-Master-Mix, ersetzt dTTP dUTP zu Amplifikationseffizienz 3) Proprietary Reaktionspuffer, optimiert, um mit Solaris Primer / Sonden-Assays zu maximieren. Diese Reaktion Puffer enthält blauen Farbstoff, um den Kontrast zwischen Reagenz und Kunststoff zu erhöhen. 4) ROX, wenn Ihr qPCR Gerät benötigt ROX. </li><li> Nachdem die Lösungen aufgetaut haben, Dazu den Schlauch (nicht vortexen) mischen. Spin sie nieder, um den Verlust von Reagenz zu verhindern. </li><li> Als nächstes bereiten ein 15 mL sterile Falcon-Röhrchen zum Einrichten der Reaktion Mix für das Gen von Interesse, plus einer für den Gen, Scaling-up in Abhängigkeit von der Anzahl der Proben. Bereiten Sie genug, so dass drei Wiederholungen kann für jede Probe vorbereitet werden, einschließlich einer Nicht-Template (NTC) und Standard-Kurve Proben, wenn die Standard-Kurve zur Analyse verwendet werden. </li><li> Für jede Probe auf einer 96-Well-Platte laufen, mischen Sie die folgenden: 5 ul des 2X Solaris qPCR Master Mix, .5 ul des Solaris Primer / Probe-Set (20X) und PCR-Wasser, so dass die Endreaktionsvolumen einmal die cDNA angefügt werden mit 25 ul werden. Für jede Probe auf einer 384-Well-Platte laufen Mix folgende: 12,5 ul des 2X Solaris qPCR Master Mix, 1,25 ul des Solaris Primer / Probe-Set (20X) und PCR-Wasser, so dass die Endreaktionsvolumen, sobald die cDNA hinzugefügt werden 10 ul werden. </li><li> Mit einer Multi-Kanal-Pipette, übertragen Sie die Master-Mix in die entsprechenden Vertiefungen der Platte. Der blaue Farbstoff in der Master-Mix enthalten wird bei der Verfolgung Pipettieren Fortschritte auf dem weißen Teller Hilfe. Dann fügen Sie von 1-5 ul cDNA-Template (für eine 96-Well-Platte) oder 1-2 ul der cDNA-Template (für eine 384-Well-Platte). Pipette auf und ab zu mischen. Die Platte wird in eine Zentrifuge und Spin-Down, um Luftblasen zu entfernen, da diese mit der Fluoreszenz-Messungen stören. </li><li> Programmieren Sie den Thermocycler: Das Enzym muss aktiviert werden, eine 95 ° C 15 min (1 Zyklus). Dann wurden 40 Zyklen von 95 ° C, Denaturierung für 15 Sekunden und Annealing und Extension bei 60 ° C für 60 Sekunden. </li><li> Die Platte wird in der qPCR Instrument. Starten Sie das Programm. </li></ol> Vertreter qPCR Ergebnisse mit Solaris <img src="/files/ftp_upload/1700/1700fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Solaris-Assays geben zuverlässige Erkennung selbst bei sehr niedrigen Konzentrationen Eingang, wie die PCR-Effizienz und r2-Werte beurteilt. Zehn 10-fache Verdünnungen der cDNA aus synthetischen RNA-Amplikon DNA-Sequenz oder Amplikon-Sequenz synthetisiert wurde auf einem ABI 7900HT Instrument mit Solaris qPCR Gene Expression Assays für F2RL1 oder Cdc20 bzw. verstärkt. Die log-Skala Amplifikationskurven und Standard-Kurven sind zusammen mit der Leistung der einzelnen Tests einschließlich Effizienz, r2 Wert, Dynamikbereich von 10 log10 Verdünnungen und die untere Nachweisgrenze dargestellt. <img src="/files/ftp_upload/1700/1700fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Solaris Assays geben konsistente Ergebnisse auch zwischen verschiedenen Forscher und Laboratorien. siRNAs ALDOA und PPIB, sowie eine Non-Targeting-Control (NTC), wurden in HeLa-Zellen bei 100, 10, 1 und 0,1 nM Endkonzentration transfiziert. Die Zellen wurden geerntet und Gesamt-RNA isoliert 48 Stunden nach der Behandlung. cDNA wurde unter Verwendung von Thermo Scientific Verso cDNA Synthesis Kit. Ein Aliquot der cDNA wurde in zwei räumlich getrennten Labors verstärkt mit Solaris qPCR Gene Expression Assays für den Nachweis von ALDOA, PPIB und GAPDH auf einem Roche LightCycler 480 (384er)-Plattform. Knockdown wurde anhand der ΔΔCq Methode (normiert auf GAPDH Referenz-Gens und NTC-behandelten Zellen). Das gleiche Maß an Knockdown wurden für beide Gen-Targets zwischen beiden Forscher an beiden Standorten unter Beweis gestellt.

Die Autoren dieses Artikels sind die Mitarbeiter von Thermo Scientific.

Thermo Scientific Solaris qPCR-Assays bieten eine einfache, verbesserte Verfahren zur Durchführung von qPCR-Analyse. Mit einer hohen Stringenz Algorithmus unter Einbeziehung MGB-Technologie und Superbases maximieren diese Primer und Sonde setzt die Treue und die Robustheit der qPCR-Experimente. Ein Primer und Sonde gesetzt erkennt alle bekannten Spleißvarianten eines bestimmten Gens. Die Ziel-Website, wo immer möglich, umfasst Exon Spanning Regionen, um sicherzustellen, dass genomische DNA nicht verstärkt wird. Diese Eigenschaften machen es möglich, zahlreiche Proben gleichzeitig ausgeführt werden, sehr zur Verringerung der Menge an Zeit benötigt, um komplexe Experimente durchzuführen. Da die Master-Mix ist blau, ist es möglich, weiß-opake Platten, die die Empfindlichkeit der qPCR zu verbessern, ohne den Überblick zu verlieren Pipettieren verwenden. Zusammengenommen machen die Design-Merkmale der Solaris qPCR Assay diesem Roman qPCR Assay eine einfache, aber leistungsfähige Test, dass sowohl spezifische als auch robust ist.

quantitative real-time pcr using the thermo scientific solaris qpcr assay

Die Solaris qPCR Gene Expression Assay ist eine neue Art von Primer / Sonden gesetzt, entwickelt, um die qPCR-Prozess zu vereinfachen und gleichzeitig die Empfindlichkeit und Genauigkeit des Assays. Diese Primer / Sonden-Sets sind zu&gt; 98% der humanen und Maus-Genom und verfügen über signifikante Verbesserungen von bereits verfügbaren Technologien pre-designed. Diese Verbesserungen waren möglich durch ein neuartiges Design-Algorithmus von Thermo Scientific Bioinformatik-Experten entwickelt. Mehrere praktische Funktionen wurden in die Solaris qPCR Assay um den Prozess der Durchführung quantitativer real-time PCR rationalisieren eingeflossen. Zuerst wird das Protokoll ähnlich üblicherweise eingesetzten Alternativen, so dass die Methoden bei der qPCR verwendet werden wahrscheinlich vertraut sein. Zweitens ist die Master-Mix Blau, das macht die Einstellung der qPCR Reaktionen leichter zu verfolgen. Drittens sind die Temperaturwechsel die gleichen Bedingungen für alle Assays (Gene), die es ermöglichen, viele Proben gleichzeitig ausgeführt und reduzieren das Potenzial für Fehler. Schließlich werden die Sonde und Primer-Sequenz zur Verfügung gestellten Informationen und vereinfacht den Publikationsprozess. Hier zeigen wir, wie Sie die entsprechenden Solaris Reagenzien mit dem GENEius Produkt Suchfunktion auf der Website zum Bestellen (www.thermo.com / Solaris) und wie die Solaris-Reagenzien für die Durchführung von qPCR mittels Standardkurve Methode gefunden zu erhalten.

Watch this Scientific Journal Video about Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay at JoVE.com

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

Die Solaris qPCR Gene Expression Assays sind neuartige pre-designed qPCR Primer / Sonden-Kombinationen entwickelt, um die qPCR-Prozess, ohne die Spezifität und Robustheit des Tests zu vereinfachen.

Quantitative Real-Time PCR unter Verwendung der 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 ...

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

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Mikrovolumen Proteinkonzentrationsbestimmung mit dem NanoDrop 2000c Spektrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Forschung

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.

Analysis of Gene Expression in Emerald Ash Borer ( Agrilus planipennis) Mittels quantitativer 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 ...

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.

Profiling von Pre-micro RNAs und microRNAs mittels quantitativer 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 ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop Mikrovolumen Quantifizierung von Nukleinsäuren

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Bestimmung genetischer Expressionsprofile in C. elegans Verwenden Microarray-und Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Genotypisierung von Pflanzen und Tieren Proben ohne vorherige DNA-Aufreinigung

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET-Mikroskopie zur Echtzeit-Überwachung der Signalisierung Events in lebenden Zellen mit unimolekularen Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

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

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Quantitative, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen Molecular Beacons

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

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Echtzeit-Messungen von Membranprotein: Rezeptor-Wechselwirkungen Mit Oberflächenplasmonenresonanz (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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.

Sonde-basierte Echtzeit-PCR Ansätze zur quantitativen Messung von 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 ...