Automation of Nucleic Acid Isolation on KingFisher Magnetic Particle Processors

Abstract

We describe automated nucleic acid (NA) isolation from diverse sample types using MagMAX kits (Ambion, Inc.) on KingFisher Magnetic Particle Processors (Thermo Scientific). The MagMAX-96 Blood RNA Isolation Kit is designed for total RNA isolation from whole blood from several species, without white blood cell fractionation, in about 45 min (including genomic DNA removal). The MagMAX-96 Total RNA Isolation Kit is designed for total RNA isolation from up to 2 × 10 6 cultured cells and up to 10-mg tissue. The isolated total RNA is highly intact and pure, ready to use in downstream applications such as microarray analysis or real-time reverse transcription (RT)-PCR for gene expression profiling or pathogen detection. The MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA and DNA isolation from cell free or nearly cell-free samples such as swabs, serum, and plasma; it takes about 15 min. Total NA of high quality and purity is recovered at >75% efficiency, providing high sensitivity for pathogen detection by real-time RT-PCR. Unlike automated liquid handling systems that move reagents into and out of a single well of a multiwell plate to perform the different steps of an RNA isolation procedure, the KingFisher Magnetic Particle Processors use permanent magnetic rods to collect magnetic beads from solution and release them into another well containing reagent for the subsequent step of the procedure. The effectiveness of bead collection and transfer lead to superior washing and elution efficiency, as well as rapid processing. It is a very effective strategy for automation of magnetic-bead-based NA isolation kits. (JALA 2007; 12:195–201)

High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

“…The use of a commercial DNA extraction procedure (Qiagen Bio-Sprint 96 one-for-all vet kit) gave high sensitivity as well as highthroughput capacity in a diagnostic laboratory setting. Magnetic bead DNA purification methods generally lead to lower carryover of contaminants or potential PCR inhibitors from a sample, as the method involves the physical removal of the DNA from the remainder of the sample (31)(32)(33)(34).…”

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

“…They are particularly suitable for automated platforms such as the BioSprint ® 96 Extraction Robotic Platform (Qiagen, Hilden, Germany), the KingFisher ® 96, and MagMAX ™ Express Magnetic Particle Processors (Thermo Fisher Scientific Inc., Waltham, MA, USA), respectively. Unlike classical DNA extraction methods, where reagents are moved into and out of a single well to perform the different steps of a DNA isolation procedure, permanent magnetic rods are used collecting paramagnetic beads to which the DNA is bound and released into 96‐well plates (Fang et al., ). Automated extractions obtain purified DNA in sufficient quality and purity and proved to be consistent and reproducible (Loeffler, Schmidt, Hebart, & Einsele, ).…”

Immiscible Phase Nucleic Acid Purification Eliminates PCR Inhibitors with a Single Pass of Paramagnetic Particles through a Hydrophobic Liquid

“…Alternatively, systems have been developed recently to move PMPs to a new well with magnetic probes. 17,18 However, repeated washing is still required because interferents carry over in liquid entrapped in the pellet and in thin films on the probes themselves.…”