Reinstwasser als Bestandteil multi-parametrischer Messverfahren in der Wirkstoffforschung
Originale
Key Words Reinstwasser | EXTassays | multi-parametrische zellbasierte Assays | Profilierung zellulärer Signale
Abstract
Ultrapure water as a component of multi-parametric assays used in drug discovery
Applications for ultrapure water are wide-spread within biotechnology, as it is used for cell culture, biochemistry, and molecular biology, such as polymerase chain reactions (PCR). Here, the specific use of ultrapure water generated from the arium® pro VF device is described as an integral component of cell-based multi-parametric profiling assays. These assays, termed EXTassays, are used at early stages of drug discovery. Within these assays, cellular signaling events are captured by molecularly barcoded reporters, resulting in the acquisition of large data sets, which are obtained within one measurement using Next Generation Sequencing (NGS). In a sample experimental setup, differential signalling properties were addressed, which were either caused by a specific stimulus (EGF-like domain) or a broad stimulus (PMA and serum). EGF-like domain-treated cells displayed an immediate early gene response, whereas the addition of PMA and serum caused an activation of immune response pathways. Addition of lapatinib inhibited virtually all responses induced by EGF-like domain, whereas PMA/serum-mediated responses were only partially reverted. Notably, ultrapure water was effectively used in various processing steps, which, amongst others, included the isolation of molecular barcode reporters, their amplification using various PCR strategies, and the NGS run. Taken together, the ultrapure water applied meets all the criteria to be used in sensitive applications of molecular biology.
· 2Sartorius Lab Instruments GmbH & Co. KG, Göttingen
Korrespondenz:
Michael C. Wehr, Systasy Bioscience GmbH, Adams-Lehmann-Str. 56, 80797 München; e-mail: wehr@systasy.de
Zusammenfassung
Reinstwasser wird für verschiedene molekular- und zellbiologische Anwendungen in der Biotechnologie eingesetzt, wie z. B. in der Polymerase-Ketten-Reaktion (PCR). Weiterhin findet Reinstwasser als Bestandteil multiparametrischer zellbasierter Assays Anwendung. Diese Assays, die EXTassays genannt und in der
