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Matching Grinding Method to Sample TypeOptimizing biological molecule extraction from brittle or hypocellular biological materialsby Erika Lapinskas, Ph.D., Sartorius Stedim Biotech
 Hierarchy of homogenization equipment best suited for use on hard or brittle biological materials for biological molecule extraction.
Click to enlarge. | A dual problem exists when attempting to study the cellular components of structural tissues. Hard and/or brittle materials of biological origin (bone, hair, teeth, wood etc.) typically contain a low number of cells from which biological materials of interest, such as RNA, protein and DNA, can be extracted. The high proportion of structural or storage material in such samples, like collagen, lignin, keratin, starch or a calcified matrix, poses a barrier to the solubilization of the cells in extraction reagents and also to effective disruption and homogenization of such materials.
A variety of methods for efficient extraction of RNA, DNA, protein and cell metabolites are well established and reagents are commercially available from many life science companies. However, these methodologies are least effective when used on hard/brittle biological samples unless disruption and homogenization of the sample is also optimal. Lack of consistency of homogenization of such samples may reasonably be assumed to result in variable and non-reproducible results between laboratories where differing and/or inadequate homogenization techniques have been utilized.1 These issues also apply to hard but more highly cellular samples, such as fresh-frozen tissue used for quantitative analysis of tissue-associated biomarkers.2
Typically, research and diagnostic laboratories have access to small quantities of tissue (approx. 50 to 500 mg), and utilize pre-existing equipment to homogenize their samples—rotor stator homogenizers, blade homogenizers or even direct application of an enzymatic homogenization solution to the sample. These techniques differ in effectiveness depending on the sample (see table).
Several studies utilizing hypocellular plant and animal tissues have recommended the generation of a frozen powder prior to addition of an RNA or protein extraction buffer and have demonstrated improved quality and quantity of extract.3-5 Each utilized a different grinding methodology (kitchen coffee grinder, mortar and pestle, laboratory ball mill) and while results were all improved, the laboratory ball mill has multiple inherent advantages over other grinding strategies.
In 2007, for the PathoBiology Group of the European Organisation for Research and Treatment of Cancer (EORTC), M. Schmitt and his peers published a standard operating protocol for the extraction of protein from fresh-frozen tumor tissue for the purpose of analysis of tissue-associated biomarkers. They recommended a laboratory ball mill with a 9-mm steel grinding ball and disposable 1.2-mL cryotubes to create a deep-frozen powder and then continue RNA, protein and DNA extraction by standard methods. The method utilizing laboratory ball mill was advantageous because it maintained the deep frozen state of the tissue utilizing a 30-second grinding time, pulverized effectively to allow penetration of extraction reagents, and did not require cleaning between samples. This methodology does not require the addition of enzymes or proteolytic reagents that might otherwise interfere with subsequent downstream analysis.
Different homogenization techniques each have their own optimal application, and a one-size-fits-all approach in the laboratory is not appropriate to ensure reproducible results. Any homogenization technology should be employed for the sample type and outcome for which it is best suited. For disruption of small quantities of hard/brittle/deep frozen biological materials, the most advantageous homogenization system is a laboratory ball mill.
References 1. Schmitt, M., N. Harbeck, M. G. Daidone et al. 2004. Identification, validation, and clinical implementation of tumor-associated biomarkers to improve therapy concepts, survival, and quality of life of cancer patients: Tasks of the Receptor and Biomarker Group of the European Organization for Research and Treatment of Cancer. International Journal of Oncology. 25: 1397-406.
2. Schmitt, M., K. Mengele, E. Schueren et al. 2007. European Organisation for Research and Treatment of Cancer (EORTC) Pathobiology Group standard operating procedure for the preparation of human tumour tissue extracts suited for the quantitiative analysis of tissue-associated biomarkers. European Journal of Cancer. 43: 835-844.
3. Wang, S. X., W. Hunter, and A. Plant. 2000. Isolation and purification of functional total RNA from woody branches and needles of Sitka and white spruce. BioTechniques. 28: 292-296.
4. Li, Z., and H.N. Trick. 2005. Rapid method for high-quality RNA isolation from seed endosperm containing high levels of startch. BioTechniques. 38: 872-876.
5. Reno, C., P. Marchuk, P. Sciore et al. 1997. Rapid isolation of total RNA from small samples of hypocellular, dense connective tissue. BioTechniques. 22: 1082-1086.
6. Hopkins, T. 2000. Laboratory cell disruptors: A review of apparatus and techniques. Int Lab News. (October) 16-17.
For more information, contact Erika Lapinskas, Ph.D., at erika.lapinskas@sartorius-stedim.com or by phone at 631-559-2097 or visit www.sartorius-stedim.com.
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