RNA Integrity Number (RIN) – Standardization of RNA Quality Control Application

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Using intact RNA is a key element for successful microarray or RT-PCR analyses. The Agilent 2100 bioanalyzer and RNA LabChip kits play an important role in assisting researchers in the determination of RNA quality. Profiles generated on the Agilent 2100 bioanalyzer yield information on concentration, allow a visual inspection of RNA integrity, and generate ribosomal ratios. This Application Note describes a new software algorithm that has been developed to extract information about RNA sample integrity from a bioanalyzer electrophoretic trace. Odilo Mueller Samar Lightfoot Andreas Schroeder somal peaks and the lower marker. The bioanalyzer software automatically generates the ratio of the 18S to 28S ribosomal subunits. Although ribosomal ratios play an important role in determining the level of sample degradation in gel electrophoresis, the more detailed analysis on the Agilent 2100 bioanalyzer reveals that it inadequately describes sample integrity. In order to standardize the process of RNA integrity interpretation, Agilent Technologies has introduced a new tool for RNA quality assessment. The RNA Integrity Number (RIN), was developed to remove individual interpretation in RNA quality control. It takes the entire electrophoretic trace into account. The RIN software algorithm allows for the classification of Introduction Determining the integrity of RNA starting materials is a critical step in gene expression analysis. The Agilent 2100 bioanalyzer and associated RNA 6000 Nano and Pico LabChip kits have become the standard in RNA quality assessment and quantitation. Using electrophoretic separation on microfabricated chips, RNA samples are separated and subsequently detected via laser induced fluorescence detection. The bioanalyzer software generates an electropherogram and gel-like image and displays results such as sample concentration and the socalled ribosomal ratio. The electropherogram provides a detailed visual assessment of the quality of an RNA sample. However, methods that rely on human visual interpretation of data are intrinsically flawed. Previously, researchers have used the ribosomal ratio in both slab gel analysis and as a feature within the bioanalyzer software to characterize the state of RNA intactness. Slab gel analysis of total RNA samples using ribosomal ratios often results in an inaccurate assessment of the RNA integrity. The Agilent 2100 bioanalyzer provides a better assessment of RNA intactness by showing a detailed picture of the size distribution of RNA fragments. RNA degradation is a gradual process. As degradation proceeds (figure 1), there is a decrease in the 18S to 28S ribosomal band ratio and an increase in the baseline signal between the two riboeukaryotic total RNA, based on a numbering system from 1 to 10, with 1 being the most degraded profile and 10 being the most intact. In this way, interpretation of an electropherogram is facilitated, comparison of samples is enabled and repeatability of experiments is ensured. Development of the RIN tool The RIN software algorithm was developed for samples acquired with the Eukaryote Total RNA Nano assay on the Agilent 2100 bioanalyzer. Input data included approximately 1300 total RNA samples from various tissues, three mammalian species (human, mouse and rat), all with varying levels of integrity. Categorization of the RNA samples was done manually by application specialists who classified each total RNA 2 Figure 1 A total RNA sample was degraded for varying times and the resulting samples were analyzed on the Agilent 2100 bioanalyzer using the Eukaryote Total RNA Nano assay. A shift towards shorter fragment sizes can be observed with progressing degradation. sample within a predefined numeric system from 1 through 10. Figure 2 shows representative electropherograms for different RIN classes (10, 6, 3, 2, respectively). For development of the RIN algorithm, adaptive learning tools, such as neural networks, were employed (tools provided by quantiom bioinformatics). They allowed the determination of critical features that can be extracted from an electrophoretic trace. These features are parts of an electropherogram that can be analyzed using an appropriate integrator. They can be signal areas, intensities, ratios etc. Important elements of an electropherogram are listed in figure 3. They include different regions (pre-, 5S-, fast-, inter-, precursor-, post-region) and peaks (marker, 18S, 28S). RIN visualization RIN will be part of the Agilent 2100 expert software. Data found in previous versions of the biosizing software can also be found in the next expert software version, for example, RNA area, RNA concentration, rRNA ratios. The RIN software includes the RIN number (figure 4), which can be expressed either as a decimal or integer. The RIN value can be changed from a decimal to an integer in the Assay Properties tab, in the Set Point Explorer under Global Advanced settings. RIN values may not be computed if the software finds an unexpected peak or signal in certain regions. This will result in an error message indicating that an 3 18 S 28 S Fl uo re sc en ce Time (seconds) 0 10 20 30 40 50 60 70 80 90 19 24 29 34 39 44 49 54 59 64 69 18 S 28 S Fl uo re sc en ce Time (seconds) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 24 29 34 39 44 49 54 59 64 69