Hello, I am working on circular RNA isolation and applying RNase R treatment. Although my total RNA quality is within the standard range before the reaction, I observe a significant decrease in RNA quality after RNase R incubation (15 min at 37 °C). I tested two different approaches: in one, I performed heat inactivation at 70 °C for 10 min to stop the enzyme; in the other, I directly proceeded with purification using a commercial kit without heat inactivation. In both cases, despite using the same purification kit, the RNA quality was markedly reduced. What could be the possible reason for this degradation?
Hello Safiye Özalp
RNase R has its own limitations. RNase R is known to struggle with degrading highly structured linear RNAs or those lacking a single-stranded 3' overhang needed for the enzyme to bind effectively. This can lead to incomplete removal of linear RNA, affecting the overall purity of the circular RNA isolation and leaving residual linear fragments that contaminate the sample. If you use high concentrations or if the incubation is prolonged with RNase R, it can lead to over-digestion and potential degradation of even circular RNAs, especially larger ones, possibly due to contaminating endonucleases or factors related to the specific circular RNA structure. Additionally, certain circRNAs are particularly sensitive to RNase R digestion.
To balance circRNA recovery and purity, you will have to optimize RNase R treatment conditions. You may titrate RNase R concentration and incubation time. Try to find the spot where linear RNA is effectively degraded without significantly impacting circRNA integrity. You may use lithium instead of potassium in the reaction buffer as this will aid in the degradation of linear RNAs that contain G-quadruplex structures resistant to RNase R in the presence of potassium. Try to implement methods like RPAD which stands for "RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion," that is used to isolate highly pure circular RNAs after an initial RNase R treatment. It builds upon the initial RNase R treatment by adding two additional steps to further purify the circRNA population.
Best,
Thank you for your valuable and detailed explanation. It was very helpful and much appreciated
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