RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. [1] Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. [2] [3] The filter paper based lysis and elution method features high throughput capacity. [4]
RNA extraction in liquid nitrogen, commonly using a mortar and pestle (or specialized steel devices known as tissue pulverizers) is also useful in preventing ribonuclease activity.
RNase contamination
The extraction of RNA in molecular biology experiments is greatly complicated by the presence of ubiquitous and hardy RNases that degrade RNA samples. Certain RNases can be extremely hardy and inactivating them is difficult compared to neutralizing DNases. In addition to the cellular RNases that are released there are several RNases that are present in the environment. RNases have evolved to have many extracellular functions in various organisms. [5] [6] [7] For example, RNase 7, a member of the RNase A superfamily, is secreted by human skin and serves as a potent antipathogen defence. [8] [9] For these secreted RNases, enzymatic activity may not even be necessary for the RNase's exapted function. For example, immune RNases act by destabilizing the cell membranes of bacteria. [10] [11]
To counter this, equipment used for RNA extraction is usually cleaned thoroughly, kept separate from common lab equipment and treated with various harsh chemicals that destroy RNases. For the same reason, experimenters take special care not to let their bare skin touch the equipment.
See also
References
- ^ Peirson SN, Butler JN (2007). "RNA extraction from mammalian tissues". Circadian Rhythms. Methods in Molecular Biology. Vol. 362. pp. 315–27. doi: 10.1007/978-1-59745-257-1_22. ISBN 978-1-58829-417-3. PMID 17417019.
- ^ Chomczynski P, Sacchi N (2006). "The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on". Nat Protoc. 1 (2): 581–5. doi: 10.1038/nprot.2006.83. PMID 17406285. S2CID 28653075.
- ^ Bird IM (2005). "Extraction of RNA from cells and tissue". Methods Mol. Med. 108: 139–48. doi: 10.1385/1-59259-850-1:139. ISBN 1-59259-850-1. PMID 16028681.
- ^ FortiusBio High Throughput RNA Extraction Filter Paper Card
- ^ Rossier, O.; Dao, J.; Cianciotto, N. P. (2009). "A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis". Microbiology. 155 (3): 882–890. doi: 10.1099/mic.0.023218-0. PMC 2662391. PMID 19246759.
- ^ Luhtala, N.; Parker, R. (2010). "T2 Family ribonucleases: Ancient enzymes with diverse roles". Trends in Biochemical Sciences. 35 (5): 253–259. doi: 10.1016/j.tibs.2010.02.002. PMC 2888479. PMID 20189811.
- ^ Dyer, K. D.; Rosenberg, H. F. (2006). "The RNase a superfamily: Generation of diversity and innate host defense". Molecular Diversity. 10 (4): 585–597. doi: 10.1007/s11030-006-9028-2. PMID 16969722. S2CID 20922592.
- ^ Harder, J. (2002). "RNase 7, a Novel Innate Immune Defense Antimicrobial Protein of Healthy Human Skin". Journal of Biological Chemistry. 277 (48): 46779–46784. doi: 10.1074/jbc.M207587200. PMID 12244054.
- ^ Köten, B.; Simanski, M.; Gläser, R.; Podschun, R.; Schröder, J. M.; Harder, J. R. (2009). "RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium". PLOS ONE. 4 (7): e6424. Bibcode: 2009PLoSO...4.6424K. doi: 10.1371/journal.pone.0006424. PMC 2712763. PMID 19641608.
- ^ Huang, Y. -C.; Lin, Y. -M.; Chang, T. -W.; Wu, S. -J.; Lee, Y. -S.; Chang, M. D. -T.; Chen, C.; Wu, S. -H.; Liao, Y. -D. (2006). "The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity". Journal of Biological Chemistry. 282 (7): 4626–4633. doi: 10.1074/jbc.M607321200. PMID 17150966.
- ^ Rosenberg, H. F. (2008). "RNase a ribonucleases and host defense: An evolving story". Journal of Leukocyte Biology. 83 (5): 1079–87. doi: 10.1189/jlb.1107725. PMC 2692241. PMID 18211964.
External links
Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits; by Erik Jue, Daan Witters & Rustem F. Ismagilov; Nature, Scientific reports, 2020.
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