Guide to the Disruption of Biological Samples - 2012
Part III: Harvesting Samples
Though the focus of this article is on homogenization/sample disruption, it is important to note that methods used for sample collection and harvesting can significantly impact results as well. Just as important as selecting the best homogenization method, a good harvesting method is also required. Depending upon the analyte or the component being sought, the method of collecting, harvesting, and subsequently storing should be carefully considered.
RNA and protein profiles are not uniform between cells in a heterogeneous sample. Therefore, it is important to consider whether the position of analytes within a sample is significant or whether the concentration or presence of the analyte in relative concentration to the whole sample is important. If location is important, then care needs to be exercised in not only maintaining the position of materials within the sample, but the impact of stress imparted by handling and storage should also be considered. Alternatively, homogeneous samples, such as a shaking bacterial culture, can be viewed as a uniform collection of cells.
Cultured microbes tend to be robust, unless they are highly sensitive to environmental stress (e.g., oxygen intolerance). Typically, microorganisms can be harvested and handled while chilled and subsequently processed, but this is for laboratory cultures. There is usually little concern about losing cells from a culture as it is generally assumed that all the cells are genetically identical and physiologically the same. However consideration should be given to the target molecule and its stability once the manipulation of the sample begins. For instance, E. coli cultured at 37°C in a shake flask is in a very different environment than E. coli embedded in a pellet following centrifugation. Berstein et al. (2002) reported that the half-life of approximately 80% of E. coli mRNAs are between 3 and 8 minutes, well within the time it can take to simply harvest cells from culture. Consequently, the RNA profile of a microorganism may represent the conditions of sample collection rather than culturing.
Samples collected from the environment are completely different as populations of mixed cultures can rapidly change in numbers, adhere to surfaces, and alter their metabolic profiles based on the means by which they are collected and stored. Bacteria embedded in biofilms survive based on the environmental parameters surrounding that mass and disaggregating the biofilm will certainly change the behavior of the bacteria. The physiology of Pseudomonas aeruginosa cells differ upon its position within biofilms (Pérez-Osorio et al., 2010), thus disruption during harvesting and storage may affect gene expression based on changes to microenvironments. Whether or not such changes impact the ultimate result of the collection and homogenization process needs to be considered.
What is true for bacteria is also true for other organisms. Plants certainly have periods of active metabolism where harvesting and storage conditions will impact the levels and conditions of analytes. Handling of oat seedlings, soybean hypocotyls, and potato plants all have shown changes in transcript levels based on the handling (Green, 1993). For seeds that are dried and relatively dormant, the collection and homogenization process probably has a much smaller impact on the levels and condition of analytes.
The haste by which animal tissues need to be harvested and processed is directly related to the stability of the components being sought after. DNA, RNA, proteins, and the myriad of other solutes available from biological samples are all different regarding their stability once harvested from the source. It is important that this variable is considered when designing a homogenization scheme.
For instance, human skin is a major source of collagen for biomedical devices. This skin is typically collected from cadavers well after the donor has expired as the collagen is sufficiently stable. For other proteins, this may not be the case (see link for more on the stability of proteins).
Original strategies for the isolation of RNA from animal tissues were believed to be highly dependent upon the harvesting method. Rapid harvest and freezing was believed to be critical to retaining RNA within the sample. This was followed by cryogenic grinding or homogenization under highly denaturing conditions (Kirby, 1965). It was widely taught that RNA degraded very rapidly once an organism was sacrificed. Currently, many of the original notions of the methods needed to isolate RNA impressed upon "experienced" scientists appear to be in question.
RNA is apparently not as fragile a molecule as once believed. Sharova et al. (2009) examined a wide spectrum of mouse embryonic stem cell mRNAs for their half-life and found all but a few to have a half-life of over 7 hours. This type of stability is in stark contrast to the notion that RNA turnover in cells should be measured in minutes. RNA levels in porcine retinal pigment cells (Malik et al., 2003) were relatively stable up to five hours after extracting intact eyes from sacrificed animals. Another eye opener (no pun intended) is that RNA in harvested tissues and deceased animals, including cadavers, may be relatively stable post-mortem for several hours (Lee et al., 2005), though there is significant variability between tissues and individuals. This is based upon the tissue remaining intact. Once tissues are dissected, the rate of RNA degradation increases (Fajardy et al., 2009). Generalizing, it appears that RNA can be stable for several hours in post-mortem samples as long as tissues have not been dissected or homogenized. Once homogenized, RNA is at risk of degrading.
Harvesting most biological samples involves some type of refrigeration. All molecules, including DNA, RNA, and protein, will remain intact as long as they are stored below -130°C, either in cryogenic freezers, vapor phase freezers, or submersed in liquid nitrogen (see link for more information on cryogenic storage). Many researchers routinely archive tissues in -80°C ultralow freezers, though some biological activity can exist even at this temperature.