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