Real-time polymerase chain reaction

A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively (i.e., above/below a certain amount of DNA molecules). Two common methods for the detection of PCR products in real-time PCR are (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines propose that the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR be used for reverse transcription–qPCR. The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention. Cells in all organisms regulate gene expression by turnover of gene transcripts (single stranded RNA): The amount of an expressed gene in a cell can be measured by the number of copies of an RNA transcript of that gene present in a sample. In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) with reverse transcriptase. In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers, deoxyribonucleotide triphosphates, a suitable buffer solution and a thermo-stable DNA polymerase.

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

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