(1388-A) Towards high-throughput low-cost sustainable ecotoxicity testing
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Traditional whole animal-based toxicity testing is expensive, time-consuming and unethical. Furthermore, these tests do not capture the full range of biological impacts. Thus, there is growing interest in developing alternative methods that have the potential to be less resource intensive (lower costs, quicker assays, fewer animals), while also providing more data on the mechanism of toxicity.
This poster shows how we optimized and established a high throughput in vitro transcriptomic-based toxicity testing platform for chemical and environmental risk assessment. The platform couples rainbow trout and human cell lines with QIAGEN’s UPXome ultraplex technology, so that the combined test system can also derive transcriptomic points of departure (tPODs). The advantages of this workflow are the microplate-based cell lysis, rRNA depletion and reverse transcription steps, and the incorporation of a unique sample index into the cDNA from each sample which result in a simpler workflow with reduced use of disposable plastic.
Briefly, human (Caco-2 and HepG2) and rainbow trout (RTgill-W1) cell lines were exposed for 24 h to a solvent (dimethyl sulfoxide) and positive controls (3,4-dichloroaniline, DCA – RTgill-W1 and hydrogen peroxide, H2O2 – Caco-2 and HepG2) at a series of concentrations, from which RNA libraries were prepared and sequenced using Illumina NextSeq500. Based on the sequencing data, we were able to perform transcriptomic analysis, including identifying differentially expressed genes (DEGs), calculating gene benchmark doses (BMDs), and deriving tPODs.
To streamline multiple reagent dispensing steps during rRNA depletion and reverse transcription, we adopted SPT Labtech’s mosquito® HV genomics automated pipettor. By harnessing true positive displacement technology, mosquito HV genomics can accurately handle volumes down to 500 nL, for even the most challenging liquid types. In this project, mosquito not only automated liquid handling steps but also enabled two-fold reaction miniaturization, improving workflow efficiency and daily processing throughput. The RNA-seq libraries obtained with mosquito HV genomics performed as well as or better than the full volume manually prepared libraries. Overall mapping percentage of reads and percentage of reads mapped to protein coding genes were comparable between 1X and 0.5X reactions.
Combining cell lines with the QIAGEN’s ultraplexing capabilities and mosquito HV genomics automated pipettor allowed us to perform cost-effective, large-scale gene expression studies of hundreds of genes in hundreds of samples, and subsequently to generate tPODs. This will further aid in chemical screening and prioritization in a more ethical and sustainable way.