(1018-C) Automation enablement with liquid handler and magnetic-bead based purification methods for ultra-high throughput processing of solid tumor samples in oncology research

Abstract:

Introduction: Sample preparation can be easily streamlined with bench top instruments like KingFisher™ sample purification systems such as KingFisher™ Duo Prime, KingFisher™ Flex, or KingFisher™ Apex across various research applications. Even with magnetic bead-based solutions, there is a need to support full automation with liquid handlers to remove tedious pre-processing and preparation steps before nucleic acid isolation. For workflows like sequential isolation of DNA and RNA with FFPE, MagMAX™ FFPE DNA/RNA Ultra workflows on KingFisher™ instruments with upfront deparaffinization using AutoLys M tubes, have significantly cut down time of manual processing while enabling consistency and reproducibility of sequential isolation from one single sample curl or slide. However, there is a need to prepare processing plates for two independent purification scripts on the KingFisher™ instruments. Here, we leverage the Hamilton Nimbus liquid handler with the KingFisher™ Presto system to enable full automation of sample preparation with ultra-high throughput workflows (multiple runs of 96 samples) per day using just one integrated KingFisher™ Presto system to isolate DNA and RNA sequentially using downstream oncology research workflows of NGS and dPCR.

Methods: Automated sequential extraction of DNA and RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples were isolated using the Hamilton® NIMBUS liquid handler integrated with the Kingfisher Presto system and the MagMAX™ FFPE DNA/RNA Ultra Nucleic Acid Isolation kit. To assess the performance of the automated workflow, the quality and quantity of extracted DNA and RNA were compared with those obtained using the Kingfisher Flex system. DNA and RNA concentrations were quantified using Nanodrop and Qubit spectrophotometers, respectively, while DNA and RNA quality were assessed using the Tapestation and Bioanalyzer. Functional evaluation of the extracted nucleic acids was conducted through qPCR, Ion Torrent sequencing, and digital PCR (dPCR) platform analysis.

Results: The automated nucleic acid extraction system yielding over 90% of DNA and RNA concentration in comparison to the Kingfisher Flex system. Moreover, the DNA and RNA profiles obtained from the automated system exhibited similarity to those generated using Tapestation and Bioanalyzer, indicating high-quality extraction. In qPCR targeting the GAPDH gene, the automation system displayed less than a 1 cycle difference (Cq) when compared to the Kingfisher Flex. Furthermore, Oncogenic mutation profiles were accessed using both Next-Generation Sequencing (NGS) and digital Polymerase Chain Reaction (dPCR) assays. These findings collectively validate the effectiveness of the automated nucleic acid extraction system, highlighting its potential as a valuable tool in molecular analysis workflows.