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Purify RNA Directly from Whole Blood
Dawn Obermoeller, Ambion, Inc.
•From blood to RNA in
less than an hour--no need to isolate white blood cells
•RNAlater® included
to stabilize RNA
•Samples can be
stored for days at ambient temperature
•Recovers highly
pure RNA, suitable for demanding applications
Ambion's RiboPure™-Blood procedure
overcomes the challenges involved in isolating intact RNA from
whole blood. In the past, researchers typically had to isolate
white blood cells (WBC) from whole blood prior to RNA isolation
to deal with the plethora of contaminants present in blood. One
of the challenges involved in isolating RNA from blood is that
white blood cells make up only a small fraction (about 0.2%)
of blood cells. Red blood cells, which account for the vast majority
of cells present in blood, contain large amounts of hemoglobin,
which inhibits RT-PCR. Plasma, which comprises ~60% of blood,
has a high concentration of RNases, as do some white blood cell
subtypes, making it necessary to quickly inactivate RNases in
blood samples to preserve the RNA. The large amount of hemoglobin
in whole blood further complicates RNA purification by clogging
filters, beads and other solid-phase matrices, and if carried
over will interfere with downstream applications. The RiboPure-Blood
Kit combines two sequential RNA purification techniques (Figure
1) to remove all of these potential contaminants, resulting in
high yields of pure RNA directly from whole blood (without prior
fractionation of WBCs) that is ideal for RT-PCR (Figure 2), microarrays,
or any other application with stringent purity needs.
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| Figure
1. Schematic of
RiboPure™-Blood Procedure. |
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| Figure
2. Real-time
RT-PCR from RiboPure™-Blood Isolated
RNA. Real-time
RT-PCR experiment in which p53, BTK, and GAPDH transcripts
were detected in the RNA isolated using the RiboPure-Blood
procedure. No genomic DNA was detectable in "no RT" control
reactions. |
RNA from Whole Blood in Less than One Hour
The simple RiboPure-Blood procedure
consists of three steps: 1) lysis with fresh or RNAlater-treated
whole blood in guanidinium based lysis solution, 2) initial
RNA purification by phenol/chloroform extraction, and 3) final
RNA purification on a glass fiber filter. The phenol/chloroform
extraction step removes proteins from the sample to prevent
clogging of the glass fiber filters and eliminate heme contamination.
A post-elution DNase treatment can then be performed with the
DNA-free™ Reagents included in the kit.
The entire procedure, including the optional DNase treatment,
can be completed in less than 1 hour (Figure 1).
Ideal for Rigorous Applications
By combining the phenol/chloroform extraction,
silica matrix purification, and the DNase treatment, the RiboPure-Blood
procedure removes protein, heme, genomic DNA, and RNases resulting
in high yields of pure RNA that are ideal for RT-PCR or array
analysis.
Stabilize RNA in Whole Blood
When collecting blood samples for RNA
analysis, storage of the samples prior to RNA isolation may
be necessary. The RiboPure-Blood Kit contains RNAlater for
the stabilization of RNA in whole blood. The addition of RNAlater to
blood samples stabilizes the RNA and prevents its degradation.
RNAlater treated samples can be safely stored at ambient
temperature for extended periods of time (up to several days).
As a result, samples can be safely transported from the point
of collection to a distant lab for RNA isolation without RNA
degradation.
Everything You Need
The kit provides sufficient reagents
for the stabilization and purification of RNA from 40 whole
blood samples ranging from 0.3-0.5 ml each. Average yields
of RNA are between 2-4 µg/0.5 ml of whole blood (variation
is due to donor-specific differences in WBC counts). The RNA
purified with the RiboPure-Blood Kit can be used in challenging
applications, such as RT-PCR and array analysis.
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