Quantitative RNA analysis with the FlashGel system for RNA

Introduction

Assessment of RNA quality/integrity is key to consistent performance in a variety of uses, including microarrays, RT-PCR, and RTqPCR.

Chip-based analysis methods provide a fast, sensitive, and quantitative rating of RNA quality (RNA integrity number (RIN)). However, such systems tend to be less flexible and more expensive than traditional gel-based methods of RNA analysis.

The FlashGel System is a rapid, ultra-sensitive, gel-based separation system that is compatible with image analysis packages that provide useful quantitative information regarding RNA quality as assessed by a ratio of the ribosomal RNA bands.

Methods

Degradation time course

E. coli Total RNA (Ambion) was diluted in ultra-pure water to 40 ng/ µl and subjected to degradation by incubation at 55°C for various times. Following degradation, RNA samples were prepared for analysis by combining with an equal volume of Formaldehyde Sample Buffer (Lonza), followed by denaturation at 60°C for two minutes. Aliquots containing 50 ng of total RNA were analysed by electrophoresis on a FlashGel RNA Cassette (225 V, 8 minutes, 20 minutes post-run hold prior to imaging). Cassette images were captured (Syngene ChemiGenius) and images were analysed using TotalLab TL100 (Nonlinear Dynamics) Image Analysis Software.

Comparison of replicate samples

Freshly prepared and partially degraded RNA samples (4 days at 37°C) were prepared and aliquots of RNA containing 50 ng were separated in triplicate on a FlashGel RNA Cassette. Images were captured using a Syngene ChemiGenius Imaging System and the FlashGel Camera.

Results

As can be seen in Figure 1, there is evidence of increasing RNA degradation as the incubation time is increased. The image clearly shows the disappearance of the 23S and 16S ribosomal RNA bands. The image was further examined to compare the ratio of the peak areas of the 23S and 16S ribosomal RNA bands. The data shown in Table 1 confirms increasing RNA degradation with increased incubation time, as illustrated by the reduction in the ribosomal band ratio.

The images of the freshly prepared and partially degraded RNA samples on the FlashGel RNA Cassette were captured using two different systems (Figure 2). Using the TotalLab Image Analysis Software, values for ribosomal band ratios were determined (average and standard deviation reported; Table 2). Ribosomal band ratios of replicate samples were also estimated using ImageJ Version 1.41 (NIH). As can be seen from the data presented, the ribosomal band ratios of replicate samples were very reproducible. The absolute values of ratios, however, varied depending upon image capture and image analysis methods used.

Conclusion

The FlashGel System offers fast, ultra-sensitive separation of RNA. Results are obtained in approximately 30 minutes from sample loading to image capture. Following separation, image analysis can give semi-quantitative results for as little as 25-50 ng of total RNA. Comparison of ribosomal band ratios by image analysis demonstrates decreasing values for samples with increasing levels of degradation. Consistent ribosomal band ratios were seen with multiple methods of image capture and image analysis. Absolute values of ratios seen were dependent on the specific combination of methods used. The FlashGel System offers the speed and sensitivity of a chip-based analysis system in a simpler, more flexible and less expensive format.

By Hugh White, Lonza Rockland, Inc.