PCR quantitative | EPFL Graph Search

La PCR quantitative (ou qPCR), ou PCR en temps réel, est une méthode particulière de réaction en chaîne par polymérase permettant de mesurer la quantité initiale d'ADN. En réalité, la PCR quantitative mesure le nombre d'amplicon (portion d'ADN définie par un couple d'amorces). Il existe diverses techniques permettant de quantifier l'ADN en biologie moléculaire. On peut utiliser la spectrophotométrie UV-visible à qui permet de connaître avec précision la concentration d'ADN total dans un échantillon par dosage optique. Cette méthode ne peut cependant pas permettre de connaître avec précision la quantité d'un ADN particulier (défini par une séquence précise de nucléotides). Il existe heureusement plusieurs techniques fiables permettant la quantification de l'expression d'un gène particulier comme le Southern blot, le Northern blot et bien sûr la PCR quantitative. 1985 : invention par Kary Mullis en 1985 de la technique de réaction en chaîne par polymérase (PCR). 1991 : première utilisation de sonde (sonde d’hydrolyse) en PCR publiée par Holland et collaborateurs. 1992 : première détection du produit de PCR en temps réel à l’aide d’un marquage au BET publiée par R. Higuchi et collaborateurs. La réaction en chaîne par polymérase en temps réel (PCR en temps réel) est une technique ayant de nombreuses applications, basée sur une réaction enzymatique, la PCR, et sur la mesure en continu de son produit et in vivo. Il existe différents appareils de PCR en temps réel. À chaque cycle d’amplification, la quantité d’ADN total ou d’amplicon est mesurée grâce à un marqueur fluorescent. L’obtention de la cinétique complète de la réaction de polymérisation permet d’obtenir une quantification absolue de la quantité initiale d’ADN cible, ce qui était très difficile à obtenir sans biais en PCR en point final. Le principe de la PCR en temps réel repose sur la possibilité de suivre la quantité d'ADN présente dans la réaction à tout instant et non à la fin de la PCR (PCR point final) ou au cycle (PCR semi-quantitative).

A low-cost, autonomous mobile platform for limnological investigations, supported by high-resolution mesoscale airborne imagery

David Andrew Barry, Ulrich Lemmin, Htet Kyi Wynn, Anton Ivanov, Abolfazl Irani Rahaghi, Stepan Tulyakov, Ludovic Zulliger, Nawaaz Sidharth Gujja Shaik, Jean-Luc Liardon, Philippe Olivier Paccaud, Jérôme Béguin, Beat Marcel Geissmann, Pascal Klaus

Two complementary measurement systems – built upon an autonomous floating craft and a tethered balloon – for lake research and monitoring are presented. The autonomous vehicle was assembled on a catamaran for stability, and is capable of handling a variety of instrumentation for in situ and near-surface measurements. The catamaran hulls, each equipped with a small electric motor, support rigid decks for arranging equipment. An electric generator provides full autonomy for about 8 h. The modular power supply and instrumentation data management systems are housed in two boxes, which enable rapid setup. Due to legal restrictions in Switzerland (where the craft is routinely used), the platform must be observed from an accompanying boat while in operation. Nevertheless, the control system permits fully autonomous operation, with motion controlled by speed settings and waypoints, as well as obstacle detection. On-board instrumentation is connected to a central hub for data storage, with real-time monitoring of measurements from the accompanying boat. Measurements from the floating platform are complemented by mesoscale imaging from an instrument package attached to a He-filled balloon. The aerial package records thermal and RGB imagery, and transmits it in real-time to a ground station. The balloon can be tethered to the autonomous catamaran or to the accompanying boat. Missions can be modified according to imagery and/or catamaran measurements. Illustrative results showing the surface thermal variations of Lake Geneva demonstrate the versatility of the combined floating platform/balloon imagery system setup for limnological investigations.

2019

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

The evolutionarily conserved Notch signaling cascade plays a pivotal role in the regulation of many fundamental processes such as stem cell maintenance, proliferation, and differentiation. Recently, we aimed to identify downstream target genes regulated by Notch1 during epidermal differentiation. For this purpose an Affymetrix mouse gene chip array of 14.000 genes was performed in both Notch1 gain and loss of function experiments using murine primary keratinocytes. Thirteen genes positively or negatively regulated, based on expression levels and relevance of the involved signaling pathways, were further validated by real-time PCR. Among these, we decided to focus on the Rho GTPase RhoV because it showed robust up- and down-regulation under both gain- and loss-of-function conditions. Therefore, we generated different transgenic mice expressing either a dominant active or a dominant negative form of RhoV specifically in the skin. Morphological and histological analyses did not reveal any overt skin phenotype in transgenic mice. In addition, challenging the system by performing wound healing assays demonstrated that the healing process in transgenic mice occurred normally and that the migration and/or differentiation of keratinocytes was unimpaired. Taken together, our data suggest that gain or loss of RhoV function does not perturb skin development and homeostasis, and does not affect the wound healing process. In addition to the investigation of the potential Notch target gene RhoV, we sought to generate both a floxed Jag2 gene targeted mouse line and a Notch2-specifc reporter mouse. The former was intended to enable conditional deletion of the Notch ligand Jagged2 and therefore allow its role to be studied in a loss-of-function context. The Notch2 reporter mouse is a tool enabling the in vivo study of cells that receive a Notch2-mediated signal. The strategy employs elements of a Tet-Off system and allows cells receiving a Notch2 specific signal to be identified in any tissue at any given time point.

EPFL2010

Performance of the US quench detection systems in the QUELL experiments

The Quench of Long Length (QUELL) experiments have been completed. The US contribution was to develop and implement both conventional and novel techniques in quench detection. The results of the quench detection experiments demonstrate that all US systems functioned as expected. The most important criteria for the comparison of the various quench detection systems were the time constant of the response and noise rejection by the system. The novel US internal (inside the CICC) sensors included; I) a cowound voltage sensor, and 2) a fiberoptic temperature sensor. The internal sensors combined fast response with high noise rejection capacity, and proved to offer potentially high reliability for ITER. The conventional sensors, namely the absolute pressure transducer and Venturi flowmeters, confirmed inherent thermo-hydaulic time response limitations in measurements of pressure and flow that excludes them as quench detectors for ITER and other large scale CICC applications.

1997

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

EPFL2010

A low-cost, autonomous mobile platform for limnological investigations, supported by high-resolution mesoscale airborne imagery

2019