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. 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.

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, 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).

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 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, 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 second-strand 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.