January 15, 2012 (Vol. 32, No. 2)
Miodrag Micic, Ph.D., Sc.D. vp of research and development MP Biomedicals
Jeffrey D. Whyte global product manager, molecular biology MP Biomedicals
Automated Lysis Systems Streamline Workflows
One of the most time-consuming bottlenecks in molecular biology experiments is efficient grinding, homogenization, and lysis of starting materials. This is especially important in plant research, soil, and environmental sciences, as well as for more common biomedical research areas, including extraction of DNA, RNA, and proteins from microbiological and solid tissue samples. For example, it is estimated, that in plant biology up to 20% of a lab technician’s time is spent on sample lysis.
Regardless of the nature of the research, it is essential that samples are lysed quantitatively. Equally important is that the simultaneously released macromolecules are functional and unaltered. In order to preserve fragile structures, it is often necessary to perform lysis and homogenization rapidly and at lower temperatures.
Sample Lysing Technologies
Most researchers today are using chemical, enzymatic, or low-tech mechanical sample homogenization methods. Problems surrounding these methods include inconsistent results due to operator and sample variability, the low-throughput bottleneck of a single sample-processing operation, and long processing times for hard, solid materials like plant tissues or bones.
The most common sample lysis method is grinding samples with a mortar and pestle, either at room temperature or cryogenically using liquid nitrogen. A mortar and pestle is largely unsuitable for low yield molecules and for ultrasensitive downstream detection techniques because it is virtually impossible to assure the absence of sample-to-sample cross contamination.
Other popular methods of sample preparation include ultrasonication, wherein the propagating ultrasound waves shear samples, and handheld, rotor/stator type homogenizers, both of which are limited to soft or suspended samples.
In addition to classical mechanical processes, chemical and enzymatic digestion or lysis are mainly used for cell culture and soft tissue. Examples include proteinase K or Zymolyase enzymatic lysis of mammalian tissue and yeast, however the process is time consuming, often up to 24 hours, and requires incubation at elevated temperatures. Problems arise if the molecules of interest are thermally unstable, prone to enzymatic degradation, or they are present in low abundance.
Mechanical sample lysis can be achieved in multiple ways, however, the nature of the forces acting on the sample can be one of three: cascade impaction, shearing via shear forces, and shearing by fluid vortexing. In cascade impaction, which is essentially the “hammering” of a sample with another object, the direct impact of compression induces cracks and breaking up of the sample. An example is bead beating, where the samples are exposed to the simultaneous impaction of high-density inert ceramic or metallic beads.
Shear forces work by imposing slicing motions on a sample to shred it apart, and can be applied mechanically like with safety razors or by fluid mechanics like the shear flow in a French press or in vortexer. Due to the complexity of 3-D random motion and complex velocity fields in a vortexer, the vortexing process, although strictly speaking is part of shearing, is also considered a separate milling process.
Sample Lysis Systems
The requirements for affordable sample homogenization and lysis equipment include the availability of fast, temperature-controlled processes, with complete homogenization of a variety of sample types, without extensive sample manipulations, and without the possibility of sample cross contamination or sample escape to the surrounding environment.
Now, more than ever, researchers are requiring parallelism and high throughput, i.e., the ability to process multiple samples simultaneously, and to process samples in varying volumes and sizes, in standard labware formats, and in formats amenable to downstream manual or automatic processing.
Furthermore, researchers require high reproducibility of results on identical sample types, i.e., that the same process settings produce the same quantity and quality of lysate, with minimum sample to sample variability. In many cases, temperature control is also desired.
All of these requirements can be conflicting, especially for processing difficult samples because in order to have fast and complete sample homogenization, a significant amount of energy is imparted on the samples and the released macromolecules of interest are exposed to those same milling forces and can be damaged. Therefore, it is necessary to minimize the exposure of released macromolecules by accelerating the sample-homogenization process.
Several automated sample-homogenization systems are currently available. MP Biomedicals manufacturers one such family of systems, including FastPrep-24™ and FastPrep-96™ (Figure 1).
The FastPrep systems consist of an instrument and a disposable lysing matrix, which includes standard labware ranging from 96-well plates and 2 mL tubes, up to 250 mL bottles, loaded with an optimized mixture of inert lysing matrix particles.
The lysing matrix beads are application specific, and their composition is optimized to provide fast and quantitative lysis of a selected sample type, in most cases within 40 seconds or less. The lysing beads are made of advanced ceramic materials with different hardness values, sizes, and densities, wherein the larger beads play the role of cascading impactor, and the smaller sharper beads are mainly performing lysis by shearing.
The FastPrep-24 instrument uses a unique, optimized 3-D motion to disrupt tissues and cells through the multidirectional, simultaneous beating of specialized lysing matrix beads on the sample material. The macroscopic motion of nutation results in an in-tube formation of the strong force fields, which accelerate tube content both alongside the vertical axis of the tubes, as well as in a sideways, angular motion.
This combination of force fields creates the in-tube environment into which the larger and heavier particles perform grinding homogenization and lysis by cascade impaction, and the smaller sharper particles are taken into the fluid vortex, moving in spiral or circular patterns and performing the shearing of the sample.
Concurrently, the liquid vortex, as a result of its velocity gradient differentials, performs fluid shearing by vortexing. As there are three simultaneous processes being performed in the tube on a sample, the result is a rapid lysis of even the most difficult samples like bone or plant materials within 40 seconds or less. A table of the most common lysis parameters settings for FastPrep-24 system is provided above.
The FastPrep system has optional temperature control adapters for cryogenic lysis by passive sample cooling with dry ice. Figure 2 represents RNA extracted from Cassava root, an RNase-rich plant material that is both mechanically difficult to homogenize and represents a special challenge for RNA extraction due to extra high content and activity of the RNase enzymes.
In addition to time savings, the largest benefit of an automated sample lysis system is the increased yield and quality of lysate, which assures consistency between sample preparations and eliminates operator and sample variation. Furthermore, automated lysis systems eliminate the most unpleasant repetitive manual workload and significantly improve safety by lysing potentially bio-hazardous samples within a closed, disposable environment.