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Phenol-Free RNAqueous® Technology
Ambion's RNAqueous technology
allows you to isolate RNA without the use of phenol, chloroform,
or other toxic organic chemicals. The entire procedure can be
completed in 2030 minutes (depending on the time required
for tissue disruption) and the resulting RNA is suitable for
use in most downstream applications, including Northern blots,
RT-PCR, nuclease protection assays, array probe labeling, and
in vitro translation. Ambion has incorporated the RNAqueous technology
into several RNA isolation kits, each of which has been optimized
for a specific application, sample type, or sample size.
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Ambion Kit
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Sample Size
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# of rxns
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Major Advantages
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| RNAqueous |
From 1 to 75 mg tissue or 100 to 107 cells |
50
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Basic kit for phenol-free isolation |
| RNAqueous-Midi |
100-500 mg tissue or 107 -108 cells |
15
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Basic kit for phenol-free isolation from larger samples |
| RNAqueous-4PCR |
From 1 to 75 mg tissue or 100 to 107 cells |
30
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Includes DNA-free(TM), phenol-free RNA isolation
Ideal for RT-PCR based applications |
| RNAqueous-Micro |
Up to 3 mg tissue or 1 to 105 cells |
50
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For micro-sized samples, great for LCM samples
Ideal for RT-PCR based applications, includes DNA-free(TM)
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| RNAqueous-96 |
From 0.1 to 1.5 mg tissue or 100 to
2 x 106 cells |
192
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High-throughput. 96 well plate-based RNA isolation
Includes DNase I for optional "on-filter DNase digestion |
RNAqueous-96
Automated |
From 0.1 to 1.5 mg tissue or 100 to
2 x 106 cells |
384
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Optimized for use with robots
High-throughput, 96 well plate-based RNA isolation
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Table 1. RNAqueous
Products for Different Applications.
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RNAqueous Technology
In the RNAqueous procedure, cells or
tissue are disrupted in a guanidinium thiocyanate solution.
This chaotropic agent effectively lyses cells and inactivates
endogenous ribonucleases. The lysate is then mixed with an
ethanol solution and applied to an RNA-binding glass fiber
filter (GFF). The ethanol changes the binding properties of
RNA such that it binds tightly to glass substrates. Proteins,
DNA, and other contaminants are removed in three rapid washing steps, and the
bound RNA is then eluted in a concentrated form (Figure 1). The entire procedure
can be completed in less than 20 minutes, after tissue disruption.
Technology Tailored for Your Application:
DNA-free RNA Isolation for RT-PCR
The RNAqueous-4PCR Kit
is specifically designed for RT-PCR applications and includes
reagents to remove genomic DNA that can contaminate RNA preparations
(Figure 2). The DNase treatment and removal reagents consist
of RNase-free DNase I, 10X DNase Buffer, and a DNase Removal
Reagent which allows the rapid removal of DNase without organic
extraction, heating, or precipitation. The RNAqueous-4PCR Kit
will deliver RNA that is free of genomic DNA contamination
and ready to use as input for qRT-PCR.
| Figure 1. Phenol-Free
Total RNA Isolation with Ambion's RNAqueous Technology. |
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Figure 2. RT-PCR
Experiments: Total RNA Isolated with the RNAqueous-4PCR
Kit is Free of Contaminating Genomic DNA. Total
RNA was isolated from 50 µg samples of the indicated
mouse tissues (stored in RNAlater®)
and eluted in 50 µl. Each RNA sample (µg)
was used as a template for reverse transcription and
5% of the resulting cDNA was amplified by PCR using
mouse ß-actin PCR primers. The lanes to the right
of the markers are PCR reactions performed without
reverse transcription, demonstrating the absence of
genomic DNA contamination in these RNA samples. The
lanes to the left of the markers show the ß-actin
RT-PCR product from the indicated samples.
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Micro Scale RNA Isolation
The RNAqueous-Micro
Kit is optimized for the purification of total RNA from micro-sized
samples, such as those obtained by laser capture microdissection
(LCM). Samples are instantly solubilized in a chaotropic solution
and RNA is purified on a small diameter GFF cartridge specifically
designed for small samples. The result is high quality RNA from
very small samples (1105 cells or up to 3 mg
of tissue).
High-throughput, 96 well RNA Isolation
Ambion's RNAqueous technology is also
available in a high-throughput, 96 well plate format. The RNAqueous-96
Automated Kit is ideal for the isolation of total RNA from
multiple small samples. The procedure utilizes an RNA-binding
96 well GFF plate and either vacuum filtration or centrifugation
methods to provide high yields of intact RNA. The 96 well plates
are compatible with most common 96 well plate vacuum manifolds
and 96 well plate centrifuge rotors. Alternatively, the RNAqueous
MAG-96 Automated Kit offers the same high throughput convenience
without the need for centrifugation or vacuum filtration.
The RNAqueous-96 Automated Kit and the
RNAqueous MAG-96 Automated Kit are formatted for high-throughput
RNA isolation on robotic platforms. Included in the Automated
Kit are reagents and supplies for 384 RNA isolations (4 x 96),
and software for the MultiPROBE®II HT (PerkinElmer)
and the Biomek® 2000 (Beckman Coulter) robotic
platforms.
RNA Isolation Products For All Applications
Ambion has a range of RNAqueous products
to meet your specific needs. Table 1 summarizes their uses
and major advantages. RNAqueous kits are not suitable for isolating
small RNAs such as siRNA and miRNA. For isolating small RNA
species see information on the mirVana
miRNA Isolation Kit.
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