Biomedical Engineering Reference
In-Depth Information
a size up to 150 kb. Isolation kits based on this method are commercially
available as well (QIAGEN, 2010b; Clontech, 2010).
Techniques for total RNA isolation
When isolating total RNA, it is essential to preserve integrity and purity
of isolated RNA during the entire preparation process. Any compromise
in the isolation process could signifi cantly affect RNA quality, which could
affect the accuracy of subsequent analyses, such as end-point PCR, quan-
titative real-time PCR or multiplexing, and could lead to further misin-
terpretation of the results. Since RNA is unstable and highly subjected to
enzymatic degradation when compared to DNA, extreme care should be
taken during the process from sample collection to RNA storage to ensure
high RNA quality. Common practices include using EDTA as a chelating
agent to sequester ions necessary for RNase function, storing samples in
−80°C freezers whenever possible, aseptic techniques, RNase-free labora-
tory wares, and gentle handling to avoid any possible degradation caused
by physical forces or enzymes.
RNA isolation methods include organic extraction, CsCl centrifugation,
silica-based and anion-exchange methods which are similar to those used
for DNA collection. The organic extraction method uses phenol to lyse
cells, and chloroform to separate RNA soluble in the aqueous phase from
DNA and other contaminants contained in the interface and organic phases
(Chomczynski and Sacchi, 1987, 2006). RNA extracted from the aqueous
phase is collected and recovered by alcohol precipitation. Phenol should be
inspected prior to use for the presence of a brown/yellowish discoloration
indicative of oxidation that may affect extraction results. Phenol and chloro-
form are carcinogenic which may cause potential health risks.
The CsCl centrifugation method allows for isolation of high-quality RNA
by separating RNA from other cellular components, through a long and
high-speed centrifugation (14-20 h at 350 000×
g
- 500 000×
g
) (Glisin
et al.
,
1974). The process is time-consuming, labor-intensive and not suitable for
isolation of small RNA species, such as rRNAs and tRNAs (McGarvey
et al
., 2003).
The silica-based methods for RNA extraction, as for DNA, are based on
the mechanism that silica selectively adsorbs nucleic acids in a solution with
a high chaotropic salt concentration (QIAGEN, 2010c). Chaotropic salts
in lysis buffer (see section 'Protein extraction and concentration measure-
ment') lyse cells and inhibit RNases, protecting RNA from undesired enzy-
matic degradation. A lysis buffer with an optimized salt concentration can
allow for selective binding of RNA onto a silica substrate while DNA and
other contaminants remain in solution. Commercially available spin col-
umn kits adopting this technique are commonly used for RNA isolation,
