Richard T. Schumacher

Improved extraction of Rhizoctonia and Pythium DNA from wheat roots and soil samples using pressure cycling technology

Soilborne pathogens are important biotic factors in yield reduction in the dryland cereal production region of the Pacific Northwest. Rhizoctonia solani AG-8, Rhizoctonia oryzae, and Pythium spp. are causal agents of root rot, bare patch, and damping-off of wheat (Triticum aestivum) and barley (Hordeum vulgare). Although these pathogens can be rapidly and specifically quantified using quantitative real-time PCR, the extraction of Rhizoctonia DNA from agricultural samples is often inconsistent, especially at low pathogen population densities. Using a novel extraction system that uses pressure cycling technology (PCT), we improved the extraction of R. solani AG-8 DNA up to 16-fold and of P. abappressorium DNA up to 2-fold from three types of agricultural soils compared with a bead beating extraction method. PCT also yielded quantifiable amounts of R. solani AG-8 and R. oryzae DNA from lyophilized wheat roots that were otherwise recalcitrant to homogenization. Furthermore, the extractions were so consistent that pathogen quantification generally could be derived from two rather than three or four replicated extracts. Because PCT is performed in a closed system and minimizes sample shearing and heating, it confers a substantial advantage over conventional extraction systems. Here, we report for the first time the application of PCT in a laboratory setting for the improved extraction and quantification of three types of soilborne pathogens in soil samples. The effectiveness of PCT for three soils suggests that it will be beneficial for other hard-to-extract pathogen samples.

Tired of the same old grind in the new genomics and proteomics era

Abstract New discoveries in life sciences depend on accurate analysis of biomolecules, which in turn depends on the extraction of high-quality molecules in high quantities from the tissues of plants, animals or microorganisms. The extraction process for hard-to-lyse cells and tissues has been a bottleneck in the path to discovery for many years. This review describes extraction methods currently in use, and compares them to a newly developed, automated process involving patented pressure cycling technology (PCT). The PCT sample preparation system (SPS) uses an instrument capable of rapid, temperature-controlled pressure cycling between ambient and high pressures, and single-use sample tubes containing a ram and a lysis disk. The quality and quantity of nucleic acid and protein prepared by the PCT SPS method are comparable to the older methods, whereas ease and safety of processing, reproducibility, speed and control are enhanced.

Isolation of mitochondria from cell cultures by PCT for proteomic analysis.

Proteomic profiling of mitochondria has the potential to provide insights into mitochondrial functions associated with aging, various metabolic states, and diseases such as cancer, diabetes, and cardiovascular disease [1]. Rapid and reproducible isolation of intact mitochondria is crucial for efficient enrichment and subsequent proteomic analysis of low-abundance mitochondrial proteins [2]. Here we describe a system for the isolation of intact mitochondria from rat PC12 cells using pressure cycling technology (PCT).