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Tutorials : Dec 1, 2009 ( )
Pressure Cycling for Sample Preparation
PCT System Provides Automated Alternative to Manual Methods!--h2>
Biological samples are highly heterogeneous, diverse, and complex. Virtually all analysis requires some type of sample preparation, yet analytical scientists often overlook the importance of this step. Ideally, sample preparation should be straightforward, reproducible, and capable of releasing all relevant analytes while preserving their structural integrity and biological activity.
Conventional biological sample preparation methods include mortar and pestle grinding, sonication, rotor-stator homogenization, French press, bead beating, freezer milling, enzymatic digestion, and chemical dissolution. All suffer from variability due to the method itself or operator inconsistencies. Moreover, they can result in destruction or incomplete release of analytes.
Pressure cycling technology (PCT) uses alternating cycles of hydrostatic pressure between ambient and ultrahigh levels (approximately 35,000 psi) to release target molecules safely and reproducibly from a wide variety of biological samples.
A PCT system consists of a Barocycler™ instrument, which generates alternating levels of hydrostatic pressure, and single-use sample processing containers. Pressure, pressure chamber temperature, and duration are under operator control. Pressure BioSciences has developed several kits for specific sample types and downstream analysis applications.
PCT is compatible with a wide array of reagents for the extraction of analytes of interest. For proteins or nucleic acid extraction from animal tissue, 50–150 mg of sample is placed inside a Pulse tube and subjected to a suitable pressure-cycling regimen. For tough or fibrous tissue such as animal muscle or plant leaves the sample may be pretreated with the PCT shredder directly in the tube. This step rapidly grinds the sample to increase surface area and improve extraction efficiency.
The extraction or lysis reagent is then added, and the sealed Pulse tube is placed into the Barocycler pressure chamber. During the PCT process, the sample is repeatedly pressurized and depressurized to lyse the cells. In addition, the moving ram of the Pulse tube repeatedly forces the solid tissue through the stationary lysis disk, further facilitating tissue disruption and cell lysis.
Most PCT runs for nucleic acid or protein extraction require about 10 minutes, however some difficult to extract samples may require up to 30 minutes or more for efficient extraction. After the PCT process, the Pulse tube is removed from the pressure chamber, and the sample is transferred to a centrifuge tube for further processing or analysis.
PCT’s initial target areas are genomics and proteomics, as well as lipidomics and small molecule analysis. Proteomics presents a unique situation for analytical scientists. The approximately one million potentially relevant components of the human proteome exist at a concentration dynamic range estimated at 1014. These numbers represent extremes that, even under ideal sample-preparation conditions, push the capabilities of modern instrumentation to its limits.
The results illustrated in Figure 1 show a comparison between PCT and two commonly used sample-preparation techniques for protein extraction from mouse liver lysates. PCT uncovered 200 and 175 more proteins than sonication and ground-glass tissue grinding, respectively. For this sample, PCT combines the advantages of grinding and sonication, providing several higher molecular weight spots than sonication, and more low molecular weight spots than grinding.
Similarly, a proteomic analysis of C. elegans found that PCT produced more protein spots and more higher molecular weight proteins than sonication (Figure 2).
DNA was extracted from fresh spinach leaves in DNAzol (Invitrogen, part of Life Technologies) by PCT and by a standard bead-beater protocol. Prior to pressure cycling, spinach was ground for 20 seconds in the PCT shredder. PCT extraction was performed for 30 cycles at 35,000 psi. Bead-beater extraction was performed in a bead beater for 10 10-second pulses with 1 mm zirconia beads.
Since bead beating generates a large amount of heat, samples were placed on ice between bead-beating pulses to prevent overheating. Following sample disruption by PCT or bead beating, DNA was extracted from the DNAzol as recommended by the manufacturer.
After DNA purification, triplicate samples were analyzed by electrophoresis. Analysis by gel electrophoresis shows that bead beater-extracted DNA is highly sheared, while PCT-extracted genomic DNA exhibits much less shearing (Figure 3). After extraction by PCT or bead beating, DNA was purified using the DNAzol isolation protocol for plants according to manufacturer’s instructions.
The life sciences are increasingly becoming quantitative disciplines that demand a high level of standardization lacking in conventional sample-preparation methods. Traditional sample-preparation methods are generally manual, time consuming, highly variable, and costly in terms of lost opportunity.
PCT provides rapid, automated, reproducible, and versatile extraction of molecules of interest from almost any sample type, allows the user to exquisitely control the test parameters, and is consequently a method that can effectively bring standardization to sample preparation.
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