Ambion TechNotes 11(1): Assessing RNA Quality
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Technical Resources > Reading Room > TechNotes > Volume 11:1
TechNotes 11(1)
Assessing RNA Quality
TechNotes Archive
Melanie Palmer and Ellen Prediger
Related Links:
This is the first in a series of columns on RNA quality and RNA sample assessment.
Watch for future articles on this subject in upcoming TechNotes issues.
mRNA Integrity
Effect of Freeze-Thawing of Tissue
on RNA Integrity
[read]
Make Time Stand Still with
RNAlater™
[read]
Because mRNA comprises only 1-3% of total RNA samples it is not readily
detectable even with the most sensitive of methods. Ribosomal RNA, on the
other hand, makes up >80% of total RNA samples, with the majority of that
comprised by the 28S and 18S rRNA species (in mammalian systems). mRNA
quality has historically been assessed by electrophoresis of total RNA
followed by staining with ethidium bromide (see Denaturing gel
electrophoresis at right). This method relies on the assumption that rRNA
quality and quantity reflect that of the underlying mRNA population. Because
mammalian 28S and 18S rRNAs are approximately 5 kb and 2 kb in size, the
theoretical 28S:18S ratio is approximately 2.7:1; but a 2:1 ratio has long
been considered the benchmark for intact RNA. While crisp 28S and 18S
rRNA bands are indicative of intact RNA, it is less clear how these long-lived
and abundant molecules actually reflect the quality of the underlying mRNA
population, which turns over much more rapidly.
Denaturing gel
electrophoresis. Denaturing
Visual assessment of the 28S:18S rRNA ratio on agarose gels is somewhat
subjective because appearance of rRNA bands is affected by electrophoresis
conditions, amount of RNA loaded, and saturation of ethidium bromide
fluorescence (Figure 1). An improved analytical tool for total RNA analysis is
the Agilent 2100 bioanalyzer, which uses a combination of microfluidics,
capillary electrophoresis, and fluorescence to evaluate both RNA
concentration and integrity (see Agilent 2100 bioanalyzer, right, and
www.ambion.com/prod/RNA6000 for more details about this analysis
tool). Another advantage is that it requires very small inputs, allowing the
user to assay RNA quality in limiting samples. At Ambion, we use this tool to
assess the quality of our pre-made RNAs. We have also used it to examine
the relationship between total RNA profiles and the integrity of mRNA. Some
of our results are discussed in the following sections.
Agilent 2100 bioanalyzer.
Thaw Frozen Tissues without
Damaging RNA
[read]
agarose gel systems include either
formaldehyde and MOPs buffer, or
glyoxal in the loading buffer, to
denature the RNA so that molecules
will run by size. The 28S and 18S
rRNA bands are visualized by
ethidium bromide staining. It is
typically necessary to load at least 1
µg of total RNA to visualize the rRNA
bands clearly with EtBr. More
sensitive dyes such as RiboGreen®
allow one to start with about 10X
less total RNA.
The Agilent 2100 bioanalyzer uses a
combination of microfluidics,
capillary electrophoresis, and
fluorescent dyes that bind to nucleic
acid to simultaneously evaluate both
RNA concentration and integrity. As
RNA moves through the separation
channel of the LabChip, intercalating
dye within the sieving matrix binds
the RNA and the fluorescence of
these molecules is measured as they
pass the detector. The output is a
scan of mass vs. size (Figure 1).
The 28S:18S rRNA ratio is calculated
by integrating the areas of 18S and
28S rRNA peaks and then dividing
the area of the 18S rRNA peak into
the area of the 28S rRNA peak. As
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an advantage over EtBr staining of
nucleic acid during gel
electrophoresis, the RNA6000 Nano
assay has a linear range between
50-250 ng of RNA, requiring little
input RNA. The recent introduction of
an RNA6000 pico assay allows users
to evaluate as little as 200 pg of
RNA. Because the RNA6000 assays
are non-denaturing, secondary
structure of the 28S rRNA results in
altered migration. (Note that 28S
rRNA does not migrate according to
its molecular weight (~5kb) but
rather migrates ahead of the 4 kb
size marker.)
Figure 1. RNA Expression Profiles from Different Tissues. Denaturing
agarose gel (inset) and Agilent bioanalyzer scan of Human Heart Total RNA (100
ng) (A) and HeLa cell line total RNA (B) isolated by multistep phenol extraction
and glass fiber filter binding, respectively. The heart sample had a 28S:18S rRNA
ratio of 1.51, and the HeLa cell sample had a 28S:18S rRNA ratio of 1.72.
The 28S:18S rRNA Ratio of 2 -- Is It Important?
rRNA Processing
With the exception of RNA prepared from cultured cells, it is rare to see total
RNAs that actually have a 28S:18S rRNA ratio of 2.0 or greater when
measured on the Agilent bioanalyzer (Figures 1,2,3). Ambion believes that
this is in part linked to instability of the 28S rRNA structure relative to the
18S RNA. This instability may result from its size as well as its high degree of
secondary and tertiary structure. In fact, some 23S and 28S rRNAs contain
an AU-rich sequence called a "hidden break" that can result in processing of
these rRNA species into two smaller RNAs. The molecular mechanism for this
type of processing is poorly understood. It is likely that similar structural
features may be responsible for the "hypersensitivity" of the mammalian 28S
rRNA relative to the 18S rRNA, resulting in 28S:18S rRNA ratios that are less
than the theoretical 2.7:1.
Figure 2 shows bioanalyzer profiles of total RNA isolated from 5 different
human prostates with progressively lower 28S:18S rRNA ratios. As the area
of the 28S rRNA peak decreases, reflecting breakdown, there is first a rise in
the baseline between the 18S and 28S rRNA and then a progressive increase
in the baseline area below the 18S rRNA that spreads as the 28S rRNA
fragments become smaller. However, in all but the most degraded sample
(panel E) the 18S rRNA peak remains fairly constant among samples,
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suggesting that this is not associated with large-scale degradation of the RNA
sample. Rather, this profile seems to result from breakdown of the 28S rRNA
relative to other RNAs. In fact, even when a sample appears to be fairly
degraded based on the 28S rRNA profile, the 18S rRNA and mRNAs may still
be fairly intact.
Figure 2. Breakdown of 28S rRNA Fragment. Agilent bioanalyzer scans of
human prostate total RNA (100 ng) isolated at different points during progressive
degradation of 28S rRNA.
RNA Degradation
Traditionally, emphasis on preserving RNA quality has been placed on
methods of tissue storage and disruption, with the goal of minimizing RNase
activity during these steps. However, the most critical factor for RNA quality
is the physiological state of the tissue at the point of removal, and to date
this issue has received little attention. Isolating RNA from human tissue
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presents challenges that are not always present in experimental animal work.
Confounding factors include the physiological state of the tissue prior to
death (referred to as the agonal state), and the post-mortem interval -- the
delay between time of death and tissue collection. In addition, there may be
additional delays before preservation, particularly in clinical settings, where
priorities for biopsy and transplant take precedence. Together, these factors
almost guarantee that human total RNA will rarely have 28S:18S rRNA ratios
of 2.0. Unfortunately, these factors are unavoidable and are rarely
considered when evaluating RNA quality.
Tissue Specific Differences in rRNA Ratios
Ambion has also found that rRNA ratios correlate, to some degree, with the
tissue of origin. This likely reflects tissue-specific responses to physiological
stress both prior to and following death. For example, lower rRNA ratios are
characteristic of some tissues, such as liver or lung, regardless of whether
the tissue is derived from mouse, rat, or human sources. Other tissues, such
as spleen, appear to be more resilient. Figure 3 shows several bioanalyzer
scans of total RNA from different human tissues demonstrating this
observation. Note that all total RNAs have a relatively low baseline, even
though the rRNA ratios vary from 1.95 to 1.2. Most profiles have small spikes
in fluorescence between 24 and 29 seconds, corresponding to the 5S rRNA
and other small RNAs. The fact that the smaller RNAs are not buried by
breakdown products suggests that the RNAs are largely intact. Northern blot
analysis of these samples using a GAPDH probe detect a sharp band at
approximately 1.4 kb, demonstrating that all of the samples contain intact
mRNA (data not shown).
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Figure. 3. Variation in Total RNA Profile Among Different Human Tissues.
Agilent bioanalyzer scan of Human Total RNA (100 ng) from the noted tissues
using large scale RNA preparations by multistep phenol extraction, followed by
LiCl precipitation, and DNase treatment and cleanup. While these RNA samples
had variable 28S:18S rRNA ratios (see individual panel descriptions), mRNA was
judged intact by Northern analysis with a probe to GAPDH (data not shown).
What Conclusions Can We Draw?
At this time there is no simple metric to predict whether mRNA is intact,
especially in limiting samples. The Agilent 2100 bioanalyzer has provided a
tool to more clearly evaluate each of the major components making up total
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RNA and to assess how they vary with source, time, and storage. However,
the relationship between rRNA profile and mRNA integrity is still unclear.
Certainly total RNA with a 28S:18S rRNA ratio of 2.0 denotes high quality.
However, it does not necessarily follow that total RNA with lower rRNA ratios
are of poor quality, and this is true for the majority of total RNAs.
Ensuring Quality of Purified RNA
To ensure that Ambion is providing its customers with the highest quality
human RNA available (FirstChoice® RNA; for more information, see
www.ambion.com/RNA), we have performed an extensive study on
human total RNA to analyze the impact of varying rRNA ratios on the
underlying mRNA. Assays include Northern blot analysis, first- and secondstrand cDNA synthesis, aRNA synthesis, test microarrays, and real-time PCR.
Our data suggest that RNA with lower 28S:18S ratios may be quite adequate
for most applications. This should at least comfort some of those scientists
who have struggled to obtain a rRNA ratio of 2.0 from a tissue that
consistently yields a ratio of 1.6. Such samples, indeed, generally yield good
aRNA amplification and Northern results.
Generally total RNAs with 28S:18S rRNA ratios >1.0 and a low baseline
between the 18S and 5S rRNA or Nano Marker are suitable for all but the
most stringent applications. Through extensive analysis we have determined
that the most critical factor in the above assays, aside from integrity, is
purity. Because most RNAs are used in downstream enzymatic applications,
residual contaminants will have the largest impact on the quality of the
resulting cDNA or aRNA. The most intact RNA will not perform well if the
sample contains residual organics, metal ions, or nucleases. To ensure that
your RNA is free of contaminants that can compromise integrity, perform a
simple stability test by incubating a small amount of RNA at 37°C for several
hours to overnight and compare it to a duplicate sample stored at -20°C. The
sample stored at 37°C should show a minimal decrease in the 28S:18S ratio
relative to the one stored at -20°C. In general, samples with greater than a
20% change in rRNA ratio over time may not perform well in downstream
applications.
RiboGreen® is a registered trademark of Molecular Probes.
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