Biotechnology & Immunology


Ultrapure Water as a Component of Multi-Parametric Assays Used in Drug Discovery


Sven P. Wichert1, Elmar Herbig2, Michael C. Wehr1 Correspondence: Michael C. Wehr, Systasy Bioscience GmbH, Adams-Lehmann-Str. 56, 80797 Munich, Germany; e-mail: wehr@systasy.de


Applications for ultrapure water are widespread in biotechnology as this water is used for cell cultivation, biochemical procedures and molecular biology techniques, such as polymerase chain reactions (PCR). This article describes the specifi c use of ultrapure water, generated by the arium® pro VF laboratory water system, as an integral component of cell-based multi-parametric profi ling assays. Such assays, termed EXTassays, are employed at early stages of drug discovery. In these assays, cellular signalling events are captured by molecularly barcoded reporters, resulting in the acquisition of large data sets, which are obtained using next-generation sequencing (NGS). In a sample experimental setup, differential signalling properties were addressed, which were either caused by a specifi c 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 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 included, inter alia, the isolation of molecular barcode reporters, their amplifi cation using various PCR strategies and the NGS run. The ultrapure water applied meets all the criteria for use in sensitive applications of molecular biology.


In biomedical research and pharmaceutical drug discovery, cell-based assays are increasingly combined with multi-parametric assay techniques to better understand highly complex disease mechanisms and the effects of compounds targeting diseases. Such assays require the implementation of methods based on molecular and cell biology, which themselves use ultrapure water as basis for performing many individual techniques, such as polymerase chain reactions, PCR, or for cultivating cells.


Cell-based assays are applied to identify differences of defi ned cellular signalling actions caused, for example, by the addition of a given compound. These differences in activity can be determined by relative changes of defi ned classes of reporter molecules (e.g., messenger RNA [mRNA], micro-RNA, proteins, etc.). Investigating the effects of biologically relevant molecules, such as chemical compounds and antibodies, on defi ned cellular activities is commonly very time-consuming, costly, and requires considerable quantities of consumables. In addition, it is important to ensure the highest purity of the solutions employed, e.g. of water. For these reasons, multi-parametric assays are becoming increasingly important.


Description of the Assay Procedure


Cell-based multi-parametric assays allow analysis of compound actions on many cellular target molecules, called targets, and profi ling of cellular signal pathways in parallel. These comprehensive analyses of complex cellular signalling activities are required to show the desired medicinal effect on targets (ON target effects) and pathways (ON pathway effects) and to simultaneously identify any undesirable side effects (OFF target/pathway effects). Such assays may also be used for uncovering new uses of approved medical drugs in so- called drug repurposing studies. Furthermore, the acquisition of large data sets from multi- parametric assays provides remarkable cost benefi ts, which can be obtained, for instance, using EXTassay technology [1].


This technology is based on molecular barcode reporters, enabling highly paralleled assays. EXT stands for Expressed Sequence Tag: EXTs are short synthetic RNA molecules with a specifi cally encoded sequence that has a length of 49 bp. Thus, EXTassay technology permits acquisition of several millions of data sets within one measurement as individual cellular signalling events captured by molecularly encoded reporters can be analysed by sequencing [1] (Figure 1a). Standard reporter gene assays allow measurements of only one or two data points per unit. Therefore, instrument-based solutions are used to acquire large data sets. By contrast, the specifi c assay system is based on reporter molecules, so-called EXT reporters, also termed EXT barcodes. Each EXT reporter, which is invariably linked to a defi ned individual signalling event, has a molecular address that enables precise analysis of the reporter’s spatial and temporal expression. Using these barcodes, many different cellular signalling activities can be simultaneously monitored, such as the activity of receptors and downstream cellular signalling events (Figure 1b).


In the multi-parametric assays discussed in this paper, next-generation sequencing (NGS) is used as a readout for functional and quantitative analysis of the various cellular activities. NGS was initially employed to sequence genomes [2]. The high-paced advancement of NGS resulted in its present use involving medical aspects, such as therapeutic diagnostics and the identifi cation of disease risk genes. The following describes the possibilities for using ultrapure water in the various stages of this multifaceted method.


Article fi rst published in Lab Asia 24.1 January/February


Figure 1: A) The specifi c assays allow simultaneous analysis of several cellular signalling activities within one measurement. Unlike standard reporter gene assays, specifi c assays provide more than 10 million data sets per experiment. B) The assays profi le compound actions on target specifi cities and cellular signalling pathways. Signalling profi les of X-different targets under Y-different conditions are simultaneously captured in one measurement (source: all fi gures provided by the authors unless otherwise specifi ed).


Materials and Methods


Uses of Ultrapure Water for Multi-parametric Assays


Ultrapure water plays a critical role in the various steps within one experiment using specifi c reporters. In our studies autoclaved ultrapure water was used, for instance, to humidify cell culture incubators to ensure microbe- and spore-free incubation of cells. Ultrapure water was also employed to determine molecular biological parameters (e.g., concentrations of DNA and RNA).


Furthermore, ultrapure water was used to prepare samples for NGS; i.e., purifi cation of EXT reporters using an RNA isolation kit, reverse-transcribing into cDNA sequences and subsequent performance of PCR steps needed to amplify the reporter material for high- throughput sequencing. Ultrapure water was also utilised in the fi nal NGS runs performed using an Ion Torrent Personal Genome Machine® (Ion PGMTM sequencer) supplied by Life Technologies (see below).


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