RNA extraction

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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 specialised motorized grinding device cell lysis device is also useful in preventing ribonuclease activity.

RNase contamination during RNA extraction[edit]

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] In these secreted RNases, the enzymatic RNase activity may not even be necessary for its new, 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[edit]

References[edit]

  1. ^ Peirson SN, Butler JN (2007). "RNA extraction from mammalian tissues". Methods Mol. Biol. Methods in Molecular Biology 362: 315–27. doi:10.1007/978-1-59745-257-1_22. ISBN 978-1-58829-417-3. PMID 17417019. 
  2. ^ 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. 
  3. ^ Bird IM (2005). "Extraction of RNA from cells and tissue". Methods Mol. Med. 108: 139–48. PMID 16028681. 
  4. ^ High Throughput RNA Extraction Filter Paper Card
  5. ^ 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. doi:10.1099/mic.0.023218-0.  edit
  6. ^ Luhtala, N.; Parker, R. (2010). "T2 Family ribonucleases: Ancient enzymes with diverse roles". Trends in Biochemical Sciences 35 (5): 253. doi:10.1016/j.tibs.2010.02.002.  edit
  7. ^ Dyer, K. D.; Rosenberg, H. F. (2006). "The RNase a superfamily: Generation of diversity and innate host defense". Molecular Diversity 10 (4): 585. doi:10.1007/s11030-006-9028-2.  edit
  8. ^ Harder, J. (2002). "RNase 7, a Novel Innate Immune Defense Antimicrobial Protein of Healthy Human Skin". Journal of Biological Chemistry 277 (48): 46779. doi:10.1074/jbc.M207587200.  edit
  9. ^ 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. doi:10.1371/journal.pone.0006424.  edit
  10. ^ 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. doi:10.1074/jbc.M607321200.  edit
  11. ^ Rosenberg, H. F. (2008). "RNase a ribonucleases and host defense: An evolving story". Journal of Leukocyte Biology 83 (5): 1079. doi:10.1189/jlb.1107725.  edit