Assessing RNA Quality
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.
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?
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.
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?
Ensuring Quality of Purified RNA
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.