|E049||500 U (50 μl)|
RNase R is an E. coli exoribonuclease which exhibits 3'-to-5' exonuclease activity, efficiently digesting nearly all linear RNA species. This enzyme does not digest circular, lariat, or double stranded RNA with short 3’ overhangs (less than seven nucleotides). As such, this enzyme is ideally suited to the study of lariat RNA produced by traditional splicing, as well as circRNAs which arise through back-splicing. By removing linear RNAs from cellular or RNA extracts, RNase R greatly facilitates the identification of circular species through RNA-sequencing. This enables researchers to probe the landscape of splicing events with greater depth.
|Unit quantity||500 U (50 μl)|
• Enriching circRNAs in biological samples
|Format||Enzyme supplied with 10X Reaction Buffer.|
|Expression System||Recombinant E. coli|
|Reaction Definition||Use 1X RNase R Reaction Buffer and incubate at 37°C.|
200 mM Tris-HCl, 1 M KCl, 1 mM MgCl2, pH 7.5
Store all components at -20 °C. Avoid repeated freeze-thaw cycles of all components to retain maximum performance. All components are stable for 1 year from the date of shipping when stored and handled properly.
|Storage Buffer||50 mM Tris-HCl (pH 7.5), 100mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% (v/v) Glycerol.|
1) If degradation is inefficient, use a slightly higher incubation temperature (40-45°C) and supplement additional enzyme partway (e.g. 0.5 µl after 1 hour) through the procedure. The higher temperature is particularly useful for degrading highly structured linear RNAs, such as rRNAs. Do not exceed 45°C or incubate over 3 hours, as this may lead to non-enzymatic RNA degradation. 2) Magnesium at concentrations of 0.1-1.0 mM is required for optimal activity. If EDTA is present, compensate by adding MgCl2 to 1.0 mM final. 3) RNase R exhibits low activity on tRNA, rRNA and other highly structured RNAs, for which the 3’ end is double stranded with a short 3’ overhang. These RNA species can stall the enzyme and result in greatly reduced activity. For best results, removal of rRNA from total RNA extracts is highly recommended.
- Wesselhoeft, R. A., Kowalski, P. S., Parker-Hale, F. C., Huang, Y., Bisaria, N., & Anderson, D. G. "RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo" Molecular cell 74(3):508-520 (2019).