James F. Eliason, Ph.D., Asterand plc
The path to identification of new and improved drugs for the treatment of human diseases often begins with the identification of novel gene targets through RNA expression analysis. Most high throughput gene expression profiling studies use high density microarrays, which require RNA preparations with purity and integrity of the highest quality. Additional methods for target identification and validation also exist, which may not require as intact RNA. This is especially valuable for gene expression studies in human tissues, as optimal conditions for the preservation of RNA integrity cannot always be met when the samples are excised surgically or from post mortem donors. Thus it is important to have reliable methods for analyzing RNA extracted from human tissues and to understand the level of RNA integrity in each sample, so that the appropriate experimental design may be employed for target evaluation.
RNA purity is generally determined spectrophotometrically. The ratio of absorbances at 260 nm and 280 nm (A260:A280) determines the degree of protein contamination and the A260:A230 ratio is used to identify any contamination by organic solvents. These ratios should be >1.8.
RNA is rapidly digested by RNase enzymes that are nearly ubiquitous, leading to formation of shorter fragments, which can confound experimental results. Thus, it is important to test the RNA integrity. This has been traditionally performed by agarose gel electrophoresis and staining the RNA either with ethidium bromide or SYBR Green dye. In the past, the ratio between the ribosomal bands (28S:18S) was viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of high quality intact RNA. Recent widespread use of the Agilent 2100 Bioanalyzer has shown that this measure is in fact a very poor indicator of RNA quality.
The Bioanalyzer provides a rapid evaluation of RNA with small quantities of material. The electropherogram generated by the instrument provides information in addition to the ratio between the ribosomal bands (see Figure 1). These include:
A) The total area under the 28S and 18S peaks combined
B) The run time for the 18S and 28S peaks
C) The presence or absence of additional peaks between the 28S and 18S peaks or between the 18S peak and the lower marker peak
Using these criteria, Asterand developed a 5 point grading system (Fig. 1), which is very similar to one developed independently by Imbeaud et al. (1). In our system, the grade of 5 indicates the highest possible quality RNA, and 1 indicates a completely degraded sample, unsuitable for rigorous RNA-based experiments. Samples rated grade 3 and above were considered to pass our RNA Quality Control evaluation.
Figure 1. Typical profiles from the Agilent Bio-analyzer 2100 representing different Asterand RNA grades.
The Agilent software now contains an algorithm to calculate an RNA Integrity Number (RIN) where a value of 10 represents the highest quality RNA (2). We have tested this algorithm in comparison to our original method and discovered that the two have a high degree of concordance. Asterand has adapted the RIN measure for our quality control process because of its ease of use, objective nature and automatic generation. Based on comparisons with our original RNA grading system, we can separate the RIN values into 3 categories.
RNA samples with RIN between 7 and 10 are the highest quality and can be used for all types of experiments (Table 1). Those with RIN values between 5 and 7 can be used for many types of experiments, such as PCR, and samples with RIN values below 5 can still be used for some gene expression profiling techniques if they are designed specifically for samples with degraded RNA. These include PCR using short amplicons of 100 bp or less or the Illumina DASL array system.
PCR assays with short regions of amplification
Highly demanding gene array assays
Table 1. RIN numbers by application
The use of alternative technologies can be important when working with human tissue samples. Human tissues often have degraded RNA at the time of collection. This is in contrast to RNA obtained from tissues collected from young, healthy animals or from cultured cells where it is reasonable to demand that all RNA samples have high integrity with RIN values greater than 8.
Effect of Sample Source on RNA quality
Because Asterand assesses the quality of all frozen tissue samples in our XpressBANK, we have a large database of RNA quality results. We have performed an analysis of the RIN values for 28,544 of these fresh frozen samples, and the data are summarized in Table 2. The samples were from both surgical recoveries, primarily cancer, and postmortem recoveries for normal tissues and chronic diseases that are not usually treated by surgery. Approximately 62% of the surgical samples have RIN values greater than 7; whereas only 32% of the postmortem samples have similar quality. The decreased RNA quality of the postmortem samples is related to the cold ischemia time, and also seems to be related to the cause of death and thus length of the agonal period.
Total Number (%)
Postmortem Number ( %)
Surgical Number (%)
Outstanding RIN ≥7
Excellent 5 ≥ RIN <7
Good RIN <5
Table 2. Distribution of RIN values by source of samples
These results indicate that in order to have 6 samples with high quality RNA from surgical recoveries, one must collect 10 cases. Similarly, 20 cases must be collected from postmortem recoveries to get the same number of the highest quality samples. Clearly, if large numbers of cases are required for a particular study, then careful selection of assay techniques that allow use of samples with a broad range of RIN values will maximize the ease with which sufficient samples can be found to complete the study. Asterand provides frozen tissue samples with a RIN value for the RNA isolated from a portion of the specimen. In this way, we provide our customers with the most information for making informed decisions about experimental methods to employ.
Asterand provides human tissue samples, RNA, DNA, serum, plasma, fresh blood, cell lines, human primary cells and tissue microarrays. We also provide fresh tissue procurement services. The RNA quality of all the samples in Asterands XpressBANK has been tested, so it is possible to select samples based on this parameter in addition to the clinical and histopathology data associated with them. In this way, the appropriate samples and technologies can be matched for studies.
Asterand Human RNA samples
1. Imbeaud, S, Graudens, E, Boulanger, V , et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res, 2005; 33: e56.
2. Schroeder, A, Mueller, O, Stocker, S , et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol, 2006; 7: 3.