Methods for Analyzing RNA Integrity

The COVID-19 pandemic marked a turning point for RNA-based therapeutics with the approval and widespread rollout of vaccines from Pfizer/BioNTech and Moderna that use messenger RNA. Although these are the best-known examples, RNA technology is also taking off for other uses. One is RNA interference (RNAi) to treat rare inherited diseases by selectively turning off the expression of faulty genes.^1,2

With RNA technology moving into the mainstream, companies increasingly need to measure the integrity and quality of RNA during the manufacturing process. “Integrity, quality, purity, and size are all interconnected and critical for RNA-based therapeutics and vaccines,” explains Quincy Mehta, Senior Manager for Capillary Electrophoresis (CE) and Consumables at SCIEX.

“The larger something is, such as an mRNA vaccine, the higher the risk of variations, and it’s essential to ensure the product is controlled,” Mehta says, adding, “We need to know if those variations are critical to product integrity. And that's where having systems and products that can analyze product quality attributes are key.”

Manual techniques for RNA integrity

Checking the integrity of RNA is essential if you’re planning experiments because RNA is easily degraded, explains Steve Siembieda, Product Marketing Director for CE products at Agilent. If you discover the RNA is degraded, “depending on the level of degradation, you can take your chances with that material or since the IVT process is straight forward and usually robust, it is better to re-transcribe the RNA and test again,” he adds.

Electrophoresis is the only technique routinely used for looking at RNA quality, Siembieda says. He adds that, although other techniques, such as high-performance liquid chromatography or polymerase chain reaction, could theoretically be used for assessing RNA quality, they would either be too slow or be unable to detect RNA degradation.

Agarose gel electrophoresis

One of the most common electrophoresis techniques for checking the integrity of RNA is agarose gel electrophoresis.^3,4 This uses a denaturing gel made from agarose, a natural polymer purified from marine kelp,^5,6 which is often precast with wells for pipetting samples.⁷ When an electrical current is passed through the gel, negatively charged RNA migrate to the positive end of the gel with smaller fragments moving faster, meaning the RNA are sorted by size.⁴

The position of the RNA can be determined by staining the sample with a fluorescent dye, typically ethidium bromide.^4,8 By illuminating the bands of fluorescent-dyed RNA with ultraviolet radiation (UV),⁴ researchers can determine whether the RNA has degraded. As Siembieda explains, “If you look at the RNA bands, there should be nice and distinct peaks, but these become less uniform when there’s degradation—there’s a shift in size, as the RNA degrades” because the RNA becomes smaller.

Polyacrylamide gel electrophoresis

Agarose gel electrophoresis is simple to carry out, which makes it usable even by high school students. However, it is lower resolution than an alternative technique called polyacrylamide gel electrophoresis (PAGE).⁷ PAGE uses polyacrylamide gel, which forms smaller pore sizes than agarose gel, meaning smaller nucleic acids can be separated out.^8,9

Polyacrylamide gel is regarded as extremely flexible and suitable for resolving RNAs that are 20 to about 600 bases long. For checking the integrity of longer RNAs (>600 bases in length), agarose gels are seen as more appropriate (due to the larger pore sizes in the gel).^10,11

Automated techniques for checking RNA integrity

Both polyacrylamide and agarose gel electrophoresis are techniques that people begin with in the lab, notes Siembieda. “They’re inexpensive, but they are 100% manual and thus people tend to move onto more automated separation methods that are more information-rich and require much less material for analysis.”

Agilent offer nine instruments suitable for automated electrophoresis. These differ by throughput, depending on whether a customer wants to analyze RNA integrity occasionally in a single sample, compared to analyzing 96 samples in parallel. They also differ by the other functions available on the instrument, such as whether a customer also wants to analyze DNA.

According to Mehta, use of existing instruments for analyzing RNA integrity is due to the recent popularity of RNA therapies. “A lot of researchers started with proteins and learned techniques there. As they plunge into this new space of next-generation therapeutics, they’re benefitting from technology they’re already familiar with.”

Like gel electrophoresis, capillary electrophoresis (CE) uses an electrical current to separate RNA fragments by their charge and size. Unlike with gel electrophoresis, however, the separation of RNA occurs in a silica capillary tube with a buffer solution inside it.^12,13 The silica capillary tends to have a natural negative charge, and the fluid flow and separation rate along the tube depend on this charge, for example, or the properties of the buffer solution.¹⁴ RNA moves along the capillary differently, depending on its size, and can be detected using UV and other methods.¹⁵

CE is more quantitative than manual/slab gel techniques and benefits from new software and analytical techniques, according to Mehta. “There’s more quantitative capability, rather than just using band intensity,” he explains.

According to Siembieda, another advantage of CE is that it can provide a more three-dimensional assessment of RNA integrity. Rather than providing a 2D band typical of slab gel methods, CE allows the amplitude of RNA to be assessed, as well. “With gel electrophoresis, you’re just looking straight down at the band and the contrast between black and white. With our instrument, it’s like looking at a city from an airplane, you can look down at the buildings to see the amount, but when you get lower to the ground you can see the height and which building is bigger and taller. From this view, you can assess how much degradation has occurred,” he says.

References

1. Albert, H. (2022) The COVID-19 pandemic has driven RNA therapeutics into the mainstream, Labiotech.eu 

2. Kimber, E. (2022) Alnylam’s RNAi therapeutic expanded by FDA in advanced primary hyperoxaluria type 1 

3. (Accessed 2022) Is Your RNA Intact? Methods to Check RNA Integrity, Thermofisher Tech Notes 

4. Steward, K. (2022) Agarose Gel Electrophoresis, How It Works and Its Uses, Technology Networks Analysis & Separations 

5. (Accessed 2022) Agarose Gel Electrophoresis of RNA, Thermofisher RNA Protocols 

6. (Accessed 2022) Agarose: Properties and Research Applications, Merck 

7. (Accessed 2022) Agarose Gel Electrophoresis for DNA, RNA, or Protein, Bio-Rad 

8. (2021) How to Interpret Agarose Gel Data: The basics, LabXchange 

9. Steward, K. (2022) Polyacrylamide Gel Electrophoresis, How It Works, Technique Variants and Its Applications,  Technology Networks Analysis & Separations

10. (2023) Nucleotide, National Human Genome Research Institute 

11. Rio, D. C et al (2010) Polyacrylamide Gel Electrophoresis of RNA. Cold Spring Harbour Protocols, 

12. Voeten, R. L. C et al (2018) Capillary Electrophoresis: Trends and Recent Advances, Analytical Chemistry, Vol. 90 (3), pp. 1464-1481

13. Sutipatanasomboon, A. (2021) Capillary Electrophoresis: Laboratory Techniques, Protocols, Science, ConductScience.com 

14. Shrestha, A. (2022) Capillary Electrophoresis: Principle and Application, Microbe Online 

15. De Jong, G. (accessed 2022) Detection in Capillary Electrophoresis – An Introduction, Wiley-VCH