RNA extraction
{{Short description|Purification of RNA from biological samples}}
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.{{cite book |author=Peirson SN, Butler JN |title=Circadian Rhythms |chapter=RNA extraction from mammalian tissues |volume=362 |pages=[https://archive.org/details/circadianrhythms00rosa/page/315 315–27] |year=2007 |pmid=17417019 |doi=10.1007/978-1-59745-257-1_22 |series=Methods in Molecular Biology |isbn=978-1-58829-417-3 |chapter-url-access=registration |chapter-url=https://archive.org/details/circadianrhythms00rosa/page/315 }} Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction.{{cite journal |author=Chomczynski P, Sacchi N |title=The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on |journal=Nat Protoc |volume=1 |issue=2 |pages=581–5 |year=2006 |pmid=17406285 |doi=10.1038/nprot.2006.83|s2cid=28653075 }}{{cite journal |author=Bird IM |title=Extraction of RNA from cells and tissue |journal=Methods Mol. Med. |volume=108 |pages=139–48 |year=2005 |doi=10.1385/1-59259-850-1:139 |pmid=16028681|isbn=1-59259-850-1 }} Usually, the phenol-chloroform solution used for RNA extraction has lower pH, this aids in separating DNA from RNA and leads to a more pure RNA preparation. The filter paper based lysis and elution method features high throughput capacity.[http://fortiusbio.com/RNA_Extraction_Card_8.html FortiusBio High Throughput RNA Extraction Filter Paper Card]
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.{{Cite journal | doi = 10.1099/mic.0.023218-0| title = A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis| year = 2009| last1 = Rossier | first1 = O.| last2 = Dao | first2 = J.| last3 = Cianciotto | first3 = N. P.| journal = Microbiology| volume = 155| issue = 3| pages = 882–890 | doi-access = free| pmid=19246759 | pmc=2662391}}{{Cite journal | doi = 10.1016/j.tibs.2010.02.002| title = T2 Family ribonucleases: Ancient enzymes with diverse roles| year = 2010| last1 = Luhtala | first1 = N. | last2 = Parker | first2 = R. | journal = Trends in Biochemical Sciences| volume = 35| issue = 5| pages = 253–259 | pmid=20189811 | pmc=2888479}}{{Cite journal | doi = 10.1007/s11030-006-9028-2| title = The RNase a superfamily: Generation of diversity and innate host defense| year = 2006| last1 = Dyer | first1 = K. D. | last2 = Rosenberg | first2 = H. F. | journal = Molecular Diversity| volume = 10| issue = 4| pages = 585–597| url = https://zenodo.org/record/1232824 | pmid = 16969722| s2cid = 20922592}} For example, RNase 7, a member of the RNase A superfamily, is secreted by human skin and serves as a potent antipathogen defence.{{Cite journal | doi = 10.1074/jbc.M207587200| title = RNase 7, a Novel Innate Immune Defense Antimicrobial Protein of Healthy Human Skin| year = 2002| last1 = Harder | first1 = J.| journal = Journal of Biological Chemistry| volume = 277| issue = 48| pages = 46779–46784| pmid=12244054| doi-access = free}}{{Cite journal | doi = 10.1371/journal.pone.0006424| title = RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium| year = 2009| last1 = Köten | first1 = B. | last2 = Simanski | first2 = M. | last3 = Gläser | first3 = R. | last4 = Podschun | first4 = R. | last5 = Schröder | first5 = J. M. | last6 = Harder | first6 = J. R. | journal = PLOS ONE| volume = 4| issue = 7| pages = e6424 | pmid=19641608 | pmc=2712763| bibcode = 2009PLoSO...4.6424K| doi-access = free}} 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.{{Cite journal | doi = 10.1074/jbc.M607321200| title = The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity| year = 2006| last1 = Huang | first1 = Y. -C. | last2 = Lin | first2 = Y. -M. | last3 = Chang | first3 = T. -W. | last4 = Wu | first4 = S. -J. | last5 = Lee | first5 = Y. -S. | last6 = Chang | first6 = M. D. -T. | last7 = Chen | first7 = C.| last8 = Wu | first8 = S. -H. | last9 = Liao | first9 = Y. -D. | journal = Journal of Biological Chemistry| volume = 282| issue = 7| pages = 4626–4633 | pmid=17150966
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. This includes solutions that are used for RNA extraction, which can be treated with chemicals such as DEPC.{{Cite web |last=Oswald |first=Dr Nick |date=2012-10-10 |title=DEPC: The Wicked Witch of RNA? |url=https://bitesizebio.com/9429/depc-the-wicked-witch-of-rna/ |access-date=2025-04-05 |website=bitesizebio.com |language=en-US}} For the same reason, experimenters take special care not to let their bare skin touch the equipment, to avoid contaminating the sample with RNAses that are present on human skin. Broad RNAse inhibitors are also commercially available and sometimes added to in vitro transcription (RNA synthesis) reactions.https://www.neb.com/en-us/products/m0314-rnase-inhibitor-murine?srsltid=AfmBOorRgG1NYgy4ThzkW6LbtJwCHqLPjtjeCLTj_sG9qN74lyRHyVpO
See also
References
{{reflist}}
External links
[https://www.nature.com/articles/s41598-020-58586-3 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.
{{Molecular Biology}}