TechNotes 11(1) Assessing RNA Quality - VHIR

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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 

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 

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. 

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. 

Related Links: 

Effect of Freeze-Thawing of Tissue 

on RNA Integrity 

[read] 

Make Time Stand Still with 

RNAlater 

[read] 

Thaw Frozen Tissues without 

Damaging RNA 

[read] 

Denaturing gel 

electrophoresis. Denaturing 

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. 

Agilent 2100 bioanalyzer. 

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



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



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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