circular RNA assay

1. RNase R cleavage assay

The nicked RNARBD or circRNARBD was heated at 65 �C for 3 min before cooled on ice. The RNase R (Epicentre, #RNR07250) was then added and incubated at 37 �C for 5 or 15 min. The reactions were stopped by adding 23 RNA loading dye (NEB, #B0363S), and
RNAs were resolved in agarose-gel electrophoresis
—— cell , 2022

To isolate circRNAs, column-purified RNA was digested with one unit of RNaseR per microgram of RNA for 60 minutes at 37 °C with shaking at 1,000 r.p.m.
—— Nature Comm , 2018

To enrich for circRNA, 20 μg of RNA was diluted in water (86 μL final volume) and then heated at 65 °C for 3 min and cooled on ice for 3 min. 20U RNase R and 10 μL of 10× RNase R buffer (Epicenter) was added, and the reaction was incubated at 37 °C for 15 min; an additional 10U RNase R was added halfway through the reaction.
——Nature Bio, 2022

Purified circular or linear RNAs were validated by RNase R treatment normally for 45 min.
——Molecular cell, 2022

2. RNase H cleavage assay

The purified circRNARBD, nicked linear RNARBD and linear precursor were incubated with RNase H (NEB, M0297L). Site-specific cleavage was performed in reactions containing 500 ng of the targeted RNAs, 50 pmol of the sense or antisense ssDNA probe and RNase H buffer in a total volume of 18 ul. After incubation at 50 ℃ for 10 min, 2 ul of RNase H was added to the reaction for 1 h at 37 ℃ .
—— cell , 2022

RNase H nicking analysis.
Splicing reactions enriched for circRNA with RNase R and then column purified were heated at 65 °C for 5 min in the presence of a DNA probe (Supplementary Data 1) at five-fold molar excess, and then annealed at room ntemperature. Reactions were treated with RNase H (New England Biolabs) in the provided reaction buffer for 15 min at 37 C. RNA was column purified after digestion.
——Nature Comm, 2018

Protocol for cleavage with RNase H (NEB #M0297)
RNase H Catalog #: M0297S , M0297L

图片.png

1. Set-up a typical reaction as follows:

   COMPONENTS	100 μl REACTION
   RNA:DNA duplex	2 µg
   RNase H Reaction Buffer(10X)	10 µl (1X)
   RNase H	1 µl (5 units)
   Nuclease-free H2O (NEB#B1500S)	up to 100 µl

2. Incubate at 37°C for 20 minutes.

3. The reaction can be stopped with addition of 1 µl of 0.5 M EDTA.

*** Note:** If targeting an RNA in a pool of RNA, we recommend titration of the enzyme to the intended substrate. Optimization may be required based on sequence, length and the type of reaction.