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New Tools for Small RNA Analysis
Non-coding small RNAs such as transfer RNAs
(tRNA), ribosomal RNAs (rRNA), small nucleolar RNAs (snoRNA),
and small nuclear RNAs (snRNA) play critical roles in a variety
of cellular processes, from regulation of mRNA translation, pre-mRNA
splicing or transcription to chromosome maintenance and gene
imprinting. In addition, two novel classes of small non-coding
RNAs (1924 nt)--referred to as small interfering RNAs (siRNAs)
and microRNAs (miRNAs)--have recently emerged as powerful regulators
of gene expression in a variety of organisms (1,2). siRNAs are
now widely used as tools for gene silencing experiments in mammalian
cells and organisms. Here we describe methods for small RNA isolation,
labeling, and analysis.
Analyzing the Expression of Small RNAs
Currently, the analysis of miRNA and
siRNA expression suffers from two technological drawbacks.
Firstly, most standard RNA isolation protocols were not designed
to efficiently recover small RNAs species. For example, glass
fiber filters, which are widely used to prepare total RNAs,
do not quantitatively recover various RNAs that are smaller
than 200 nt (such as 5S rRNA, snRNAs, snoRNAs, tRNAs, miRNAs,
and siRNAs). Commercially available total RNA preparations
do not always contain representative amounts of small RNA species
because the RNA isolation protocols have not been optimized
to recover small RNAs. A second technological challenge is
that sensitive methods for the detection of small RNAs have
not been previously available. RT-PCR, while being very sensitive,
cannot be used to amplify small RNAs since in standard RT-PCR,
the primers are about the same size as the small RNAs. As a
result, siRNAs and miRNAs have been primarily detected by Northern
analysis, a technique that is time-consuming and relatively
insensitive.
The mirVana™ System for Small
RNA Analysis
To facilitate the analysis of small
RNA expression, Ambion has developed the mirVana system,
a complete solution for the isolation and detection of small
RNAs (Figure 1). The mirVana products include:
The mirVana™ miRNA
Isolation Kit--for isolation of total RNA containing
small RNAs, or preparation of fractions enriched in small
RNAs (<200 nt).
"miRNA certified" FirstChoice® RNAs--human,
mouse, and rat total RNAs that have been verified to contain
miRNAs and other small RNA molecules
The mirVana™ miRNA
Detection Kit--for the sensitive detection and quantitation
of miRNA and other small RNA
The mirVana™ miRNA
Probe Construction Kit--for rapid synthesis of template
and RNA probes by in vitro transcription
The mirVana™ miRNA
Probe & Marker Kit--for the 5' end-labeling and purification
of RNA or DNA probes, and preparation of small RNA markers
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Figure 1. A
Fully Integrated System for Small RNA Analysis.
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Isolation of Small RNAs
There are two widely used methods for
isolating total RNAs: 1) phenol/chaotropic reagent extraction,
and 2) binding to and elution from glass fiber filters (GFF).
Ambion has determined that many
small RNAs are not retained when total RNA is extracted by
traditional GFF methods which are commonly used in most laboratories
(see poster for more information). We have, however, developed
a modified GFF-based procedure--through the use of optimized
binding and wash solutions--that provides quantitative yield
of miRNAs and other small RNAs from virtually any tissue or
cell line. A modification of this procedure can also be used
to enrich small RNAs from a total RNA population. This technology
is now available as the mirVana miRNA
Isolation Kit (patent pending).
"miRNA Certified" FirstChoice RNAs
When purchasing commercially available
purified RNAs, it is also important to consider the RNA isolation
method used to isolate the RNA. Most of Ambion's FirstChoice
Total RNAs have been validated for the presence of small RNA
molecules--and we are constantly working on optimized isolation
procedures for these and other tissue and cell line samples.
Visit our web catalog for an up-to-date list of "miRNA
certified" FirstChoice
RNAs.
Sensitive Detection of Small RNAs
Although Northern analysis can be used
to detect and quantitate small RNA, the technique is labor-intensive
and, more importantly, not very sensitive. Ambion scientists
have developed a sensitive assay that has been optimized for
small RNA quantitation--the patented mirVana miRNA Detection
Kit--which is based on solution hybridization and subsequent
RNase protection. Short radiolabeled RNA probe is simply mixed
with total RNA in an optimized hybridization buffer and all
single-stranded RNAs (e.g. unhybridized probe, mRNAs, rRNA)
are then digested by a mixture of RNase A and T1. Probe:target
RNA hybrids are quickly recovered using a patented single-step
RNase inactivation/precipitation procedure, run on a denaturing
polyacrylamide gel, and visualized by autoradiography. In this
assay, full-length and protected probes are easily distinguished
by the presence of a short sequence at the 5' or 3' end of
probes that is not complementary to the RNA target (Figure
2).
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Figure 2. The mirVana
miRNA Detection Kit for small RNA Detection
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The mirVana miRNA Detection Kit
has been used to detect
miRNAs and siRNAs in a variety of tissues and cells (see poster for more information).
The sensitivity and specificity of the assay is illustrated
in Figure 3 where miR-124 miRNA was detected only in human
brain total RNA. This experiment confirms the tissue-specific
expression of miR-124, previously determined by Northern blot
analysis using 30 µg of total RNA (3). In contrast, only
1 µg
of total RNA was used in the solution hybridization assay.
Moreover, miR-124 expression could be detected in as little
as 10 ng of total brain RNA after overnight exposure of the
gel (data not shown).
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Figure 3. Tissue-specific
expression of miR-124 miRNA. miR-124
was detected in 1 µg of the indicated FirstChoice
Total RNA from 14 different human tissues using the mirVana
miRNA Detection Kit and a 5' end-labeled RNA probe
prepared with the mirVana Probe & Marker
kit. Gel was exposed for 5 hours. |
Another application of the mirVana
miRNA Detection Kit, is the detection of two or more RNAs in
the same sample. For instance, an expressed siRNA and its target
mRNA can be monitored simultaneously. See Application:
Analysis of miRNA Recovery After Gel Purification (sidebar)
or the Ambion miRNA Resource for additional
applications of this versatile kit.
Preparation of RNA Probes
Small RNA probes for the solution hybridization
assay can be readily prepared using one of two different methods:
in vitro transcription (IVT) or 5' end labeling of an existing
RNA oligonucleotide. Depending on the probe sequence and the
radiolabeled nucleotide used, IVT probes typically result in
~2-fold greater sensitivity than 5' end labeled probes due
to their higher specific activity (Figure 4). In contrast,
5' labeled RNA probes are more easily generated as they do
not require gel purification. Ambion has developed and optimized
kits for both probe-labeling methods -- the mirVana
miRNA Probe Construction Kit for IVT and the mirVana
Probe & Marker
Kit for 5' end labeling.
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Figure 4. Comparison
Between In Vitro Transcribed (IVT) and 5' End Labeled
Probes. The expression
of miR-16 miRNA and 5S rRNA was analyzed in 0.5, 1,
2 or 4 µg of FirstChoice Total
RNA from mouse kidney included in the mirVana
miRNA Detection Kit using the indicated DNA or RNA
probes included in the mirVana miRNA
Probe Construction Kit (IVT probes) or mirVana
Probe & Marker
Kit (5' end labeled probes).
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The mirVana miRNA
Probe Construction Kit includes reagents for preparation of
IVT templates as well as reagents for the IVT reaction. All
you need to provide is an inexpensive, desalted, target-specific
DNA oligonucleotide. A short sequence cleavable by RNase A
and T1 is added to the 5' end of probe during the procedure.
Probes prepared with the kit are therefore suitable not only
for solution hybridization assays, but also for Northern blot
or in situ hybridization analysis (using nonisotopically labeled
probes).
The new mirVana Probe & Marker
Kit provides reagents for the rapid preparation and purification
of short, high specific activity 5' end-labeled RNA probes.
Such probes are widely used in a variety of assays, including
RNA structure/function studies, Northern blot and primer extension
analysis. For solution hybridization assays, the antisense
probe should carry only a few nontarget-specific nucleotides
at its 3' end. The kit can also be used to label and purify
DNA probes (see Northern Blot in Figure 4) and to prepare small
Decade™ RNA markers (150, 100, 90, 80, 70,
60, 50, 40, 30, 20, and 10 nt; see Figure 5), which are included
in the kit.
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Figure 5. miRNA
Detection after Gel Purification. miRNAs
were gel purified on a 15% denaturing polyacrylamide
gel from 150 µg of mouse lung or thymus total
RNA using standard procedures. Recovery was analyzed
with the mirVana™ miRNA Detection
Kit and autoradiography (2 hours exposure) by comparing
the amount of miR-16 miRNA in the sample before (1%
or 0.5% of the starting total RNA) and after purification
(2% or 1% of the miRNA fraction). Percentage of recovery
(59 and 53% respectively) was then determined by quantification
with a phosphorimager. mir-16 probe and Decade Markers
were prepared with the reagents included in the mirVana™ Probe & Marker
Kit. |
A Complete Solution
Ambion's mirVana system
provides the first complete solution for small RNA analysis.
The system allows for the isolation of total RNA samples that
contain small RNAs--or the preparation of RNA samples that
are enriched for small RNAs. Alternatively, "miRNA certified" total
RNAs from over 80 different human, mouse, and rat tissues can
simply be purchased. Once the RNA samples are obtained, high
specific activity radiolabeled probes can be prepared by IVT
or by 5' end-labeling, and target small RNAs be detected using
the sensitive solution hybridization assay. The mirVana
system is ideal for the detection of small RNAs molecules in
a variety of experimental settings (see sidebar: Analysis
of miRNA Recovery after Gel Purification), deciphering
miRNA expression patterns across tissues and species, or determining
siRNA expression levels in cell lines and organisms.
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