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The approach used to disrupt tissues, cells and/or matrices is dependent upon the objectives of the researcher. This might appear as an obvious statement to the experienced scientists, but to the individual just getting started in a particular line of investigation it might not be as apparent. Deciding on a disruption method should be approached in a reverse direction, i.e., the final product should dictate the tools and methods used to produce it. For instance, producing a lysate which includes active protein requires a method that avoids denaturing conditions and harsh treatment of the sample, while isolating RNA may involve the exact opposite.
For discussion purposes, the methods used for disrupting samples has been divided into chemical and physical means. This dichotomy is relatively artificial as most homogenization/disruption processes make use of a combination of mechanical and chemical methods. However, in its simplest form, disruption can be broken down as illustrated in Figure 1.
Figure 1. Simple schematic of sample disruption options. This simplified scheme does not consider that methods are normally combined during sample disruption. Mechanical homogenization normally makes use of buffers or lysis solutions just as chemical lysis normally requires that the sample contains small particles, which is normally created using a homogenizer.
In developing a good method to disrupt samples, it is necessary to consider the desired characteristics of the final homogenate/lysate and then work backwards as to which chemistries and tools will work alone, or in combination, to yield that product. To do this, knowledge of the limitations of the target molecules is necessary. For instance, if pieces of intact membrane are required, then the homogenization process must effectively destroy tissue and cells, but prevent complete obliteration of subcellular components. If active proteins are needed, especially those which are heat labile, then processes which generate heat or cause foaming should be avoided. If quantitation of an analyte is the goal, then complete liberation of that analyte is necessary, which implies a thorough dissociation of all cellular structures. In isolating RNA, great care needs to be taken to prevent omnipresent RNases from degrading the target by lysing samples under highly denaturing conditions. In the case of homogenization, the ends do justify the means, thus it is valuable to dissect the methods available and to assess both their strengths and limitations. Table 1 provides a summary of major targets and factors which should be considered during sample disruption.
Table 1. Major biochemical targets and sample processing considerations.
|
Target |
Application |
Considerations |
|
Protein |
Activity Assays/ Characterization |
Liberation of active protein is normally done under non-denaturing conditions (i.e., no use of strong detergents or chaotropes). Samples should be kept cold to avoid inactivating heat labile proteins. Proteases may be liberated during homogenization, thus protease inhibitors may be added to the sample. For further information on retaining protein activity, . |
|
Protein |
Immunoassays |
Antibodies can bind to denatured proteins readily, thus lysates prepared for immunoassays are often done using surfactants. Protein samples for Western blots are often boiled in SDS. |
|
RNA |
Quantitation/ Cloning |
RNA in intact cells and tissues can be stable. Once it is released by homogenization, RNases can rapidly degrade the target. Consequently, isolating RNA usually involves minimizing RNase activity by preparing samples under highly denaturing conditions using chaotropes and by maintaining very low temperatures during sample disruption. |
|
DNA |
Detection/ Quantitation/ Cloning |
DNA is also threatened by nucleases upon liberation from an intact sample. However, DNase is much easier to control by the addition of EDTA which chelates Mg+2, an ion necessary for DNase activity. Many DNA isolation protocols still make use of detergents and chaotropes. |
|
Drugs |
Quantitation |
Pharmacokinetic analysis of residual drugs often uses organic based extractions with acetonitrile and methanol. The major considerations when disrupting samples with such solvents is vapor pressure created from processing samples in closed containers and flammability. |
Although defining the characteristics of a homogenate is a major consideration in selecting methods for a disruption process, it is not the only one. A second major consideration is sample throughput. A researcher in a high paced work environment knows the stress of generating mass data. Indeed as budgets tighten and staffs are “right sized”, the workload of researchers increase. In these environments, sample preparation is often the bottleneck. There are many chemistries and tools available, but it is the homogenizer type which can clog a process pipeline. Many homogenizer designs have been unchanged in half a century and were designed for scientists who might process a couple of sample per week. These older homogenizers were the centerpiece of slow laborious protocols, such as grinding tissues in liquid nitrogen with mortar and pestle. This type of process may be completely adequate for many labs, but it can be very inadequate when hundreds, if not thousands of samples must be processed daily. Therefore, the level of throughput needs to be considered in designing a scheme.
Sample disruption throughput is dependent upon whether samples come into direct contact with the homogenizer during processing. With glass homogenizers, such as the Dounce, Potter-Elvehjem, and conical ground glass, and rotor-stators time is required to clean and decontaminate the homogenizer itself following sample processing. With the case of rotor-stators, this may involve disassembling the shaft. Glass homogenizers may need to be washed with detergents and then decontaminated by baking. With manual cryogenic grinding, the time required is even greater as the liquid nitrogen chilled mortar and pestle must be allowed to warm before cleaning. If manual methods are used for processing many samples, then it is necessary to have many of these homogenizers available.
Ultrasonication probes are the exception to this issue as they can be readily cleaned. As the probes are metal and cleanable, they can be washed and decontaminated with alcohol.
Homogenizers that isolate the sample from the homogenizer are usually more accommodating for high throughput sample processing. Bead beaters are effective this way as samples are in tubes and the tubes are agitated. The homogenizer itself never touches the sample. This is also true for bath sonicators where the sample tube is immersed in a bath.