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RNA Extraction Is The Purification of

RNA extraction is the process of purifying RNA from biological samples using methods to isolate it from contaminants like ribonuclease enzymes. The most common extraction method is guanidinium thiocyanate-phenol-chloroform extraction, which uses phase separation after mixing the sample with phenol, chloroform, and guanidinium thiocyanate to separate the RNA in the aqueous upper phase from DNA and proteins in the lower organic phase.

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91 views3 pages

RNA Extraction Is The Purification of

RNA extraction is the process of purifying RNA from biological samples using methods to isolate it from contaminants like ribonuclease enzymes. The most common extraction method is guanidinium thiocyanate-phenol-chloroform extraction, which uses phase separation after mixing the sample with phenol, chloroform, and guanidinium thiocyanate to separate the RNA in the aqueous upper phase from DNA and proteins in the lower organic phase.

Uploaded by

Ali Akand Asif
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOC, PDF, TXT or read online on Scribd
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RNA extraction

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.
(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)

Phenol-chloroform extraction is a liquid-liquid extraction technique in


biochemistry. It is widely used in molecular biology for isolating DNA,
RNA and protein. Equal volumes of a phenol:chloroform mixture and an
aqueous sample are mixed, forming a biphasic mixture. This method
may take longer than a column-based system such as the silica-based
purification, but has higher purity[citation needed] and the advantage of high
recovery of RNA: an RNA column is typically unsuitable for purification
of short (<200 nucleotides) RNA species, such as siRNA, miRNA and
tRNA. Column methods also shear large DNA fragments, which may or
may not be a problem depending on downstream applications.
Protocol:

This method relies on phase separation by centrifugation of a mix of the


aqueous sample and a solution containing water-saturated phenol,
chloroform and a chaotropic denaturing solution (guanidinium
thiocyanate) resulting in an upper aqueous phase and a lower organic
phase (mainly chloroform). Nearly all of the RNA is present in the
aqueous phase, while DNA and protein partition in the interphase and
organic phase, respectively. In a last step, RNA is recovered from the
aqueous phase by precipitation with 2-propanol or ethanol. DNA will be
located in the aqueous phase in the absence of guanidinium thiocyanate
and thus the technique can be used for DNA purification alone.

Guanidinium thiocyanate denatures proteins, including RNases, and


separates rRNA from ribosomes, while phenol, isopropanol and water
are solvents with poor solubility. In the presence of chloroform or BCP
(bromochloropropane), these solvents separate entirely into two phases
that are recognized by their color: a clear, upper aqueous phase
(containing the nucleic acids) and a bright pink lower phase (containing
the proteins dissolved in phenol and the lipids dissolved in chloroform).
Other denaturing chemicals such as 2-mercaptoethanol and sarcosine
may also be used. The major downside is that Phenol and chloroform are
both hazardous and inconvenient materials, and the extraction is often
more laborious, so in recent years many companies now offer alternative
ways to isolate DNA.
Reagents
 Phenol: The phenol used for biochemistry comes as a water-
saturated solution with Tris buffer, as a Tris-buffered 50% phenol,
50% chloroform solution, or as a Tris-buffered 50% phenol, 48%
chloroform, 2% isoamyl alcohol solution (sometimes called
"25:24:1"). Phenol is naturally somewhat water-soluble, and gives
a fuzzy interface, which is sharpened by the presence of
chloroform, and the isoamyl alcohol reduces foaming. Most
solutions also have an antioxidant, as oxidized phenol damages
the nucleic acids. For RNA purification, the pH is kept around pH
4, which retains RNA in the aqueous phase preferentially. For DNA
purification, the pH is usually near 7, at which point all nucleic
acids are found in the aqueous phase.

 Chloroform: Chloroform is stabilized with small quantities of


amylene or ethanol, because exposure of pure chloroform to
oxygen and ultraviolet light produces phosgene gas. Some
chloroform solutions come as pre-made a 96% chloroform, 4%
isoamyl alcohol mixtures that can be mixed with an equal volume
of phenol to obtain the 25:24:1 solution.

 Isoamyl alcohol: Some protocols include isoamyl alcohol as a


stabilizing agent, while others do not require it at all.

Commercial reagents: TRI Reagent (Sigma-Aldrich), TRIzol (Invitrogen), Trisure (Bioline)

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