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Guide to the Disruption of Biological Samples - 2012

Table of Contents         Complete PDF

Part X: Summary & References

Summary

Effective disruption of biological samples is a process that starts with sample collection and proceeds through the homogenization process.  To generate a homogenate that is suitable, it is prudent to define the characteristics required in the final product and then choose the best method or combination of methods that will produce that product. 

Chemical and mechanical methods, by themselves, can be used to disrupt samples, but normally are used in some combination to achieve a desired homogenate.  At a minimum, buffers, chaotropes, and surfactants are common additives that help to solubilize and maintain biomolecules released during mechanical processing.  For the isolation of biomolecules which are labile or sensitive to degradation, protective chemistries partnered with a mechanical method are necessary to obtain quality homogenates.

All methods used to disrupt samples have strengths and weaknesses. Lower throughput methods can be used to process larger samples with good results, and many are economical.  High throughput methods are also effective, but the initial investment will be greater.

Many tools when used alone to homogenize samples are not as effective at disrupting samples but when combined with each other can be very useful.  Ultrasonication is the classic example as it is a poor method when used on solid samples, such as muscle, but yields the best results when combined with an early processing step.  Many of the shearing methods produce fine homogenates, but fail to disrupt all tissues.  They can be valuable in producing lysates with intact subcellular components.  Bead beating with high throughput homogenizers can be a very effective one-step homogenization method.

References

Bernstein, J.A., A.B. Khodursky, P-H. Lin, S. Lin-Chao, and S.N. Cohen.  2002.  Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays.  PNAS 99(15): 9697-9702.

Fajardy, I., E. Moitrot, A. Vambergue, M. Vandersippe-Millot, P. Deruelle and J. Rousseaux.  2009.  Time course analysis of RNA stability in human placenta.  BMC Molecular Biology 2009, 10:21

Green, P.J. 1993. Control of mRNA stability in higher plants. Plant Physiol. 102(4): 1065-1070.

Kirby, K.S.  1965.  Isolation and characterization of ribosomal ribonucleic acid.  Biochem. J. 96: 266-269.

Lee J., A. Hever, D. Willhite, A. Zlotnik, and P. Hevezi.  2005.  Effects of RNA degradation on gene expression analysis of human postmortem tissues. FASEB Journal 10.1096/fj.04-3552fje. Published online June 13, 2005.

Malik, K., C. Chen, and T. Olsen.  2003.  Stability of RNA from the Retina and Retinal Pigment Epithelium in a Porcine Model Simulating Human Eye Bank Conditions.  IOVS 44(6): 2730-2735.

Pérez-Osorio, A., K. Williamson, and M. Franklin.  2010.  Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRHA Gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection.  J. Bact. 192:2991-3000.

Sharova, L., A. Sharov, T. Nedorezov, Y. Piao, N. Shaik, and M. Ko.  2009.  Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells.  DNA Research 16, 45-58.