New England Biolabs, M0100S, RNase R

Related Categories RNases,, RNA Modification Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) RNase Inhibitor, Murine (NEB #M0314) (optional) Unit Definition One unit is defined as the amount of enzyme required to convert 75 pmoles of 20-nucleotide single-stranded RNA sequence downstream of a 38-nucleotide DNA hairpin into acid soluble ribonucleotides in a total reaction volume of 20 μl in 15 minutes at 25°C. Reaction Conditions 1X RNase R Reaction Buffer Incubate at 37°C 1X RNase R Reaction Buffer 20 mM Tris-HCl 100 mM KCl 0.1 mM MgCl2 Storage Buffer 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT surfactant 50% Glycerol pH 7.4 @ 25°C FAQ Q: Is RNase R (NEB #M0100) active at temperatures other than 37°C? A: Yes, RNase R is highly active up to ~50°C. However, incubation of RNAs at high temperatures can lead to unwanted RNA hydrolysis. RNase R activity is reduced at temperatures below 37°C. Q: Is it necessary to clean up RNA samples before RNase R (NEB #M0100) digestion? A: Yes, it is necessary to clean up RNA samples before RNase R digestion, especially if the sample contains a high concentration of EDTA. RNase R is a magnesium-dependent exoribonuclease. We recommend that RNA samples contain less than 0.1 mM EDTA before RNase R treatment. Q: How long does the 3´ single-stranded RNA overhang need to be for RNase R (NEB #M0100) to bind? A: RNase R prefers 3´ single-stranded RNA overhangs at least 10 nt in length. Longer overhangs, such as poly(A) tails, tend to result in higher RNase R activity. RNase R may not bind to overhangs that are shorter than 10 nt. Q: Can RNase R (NEB #M0100) be heat-inactivated? A: Yes, RNase R can be heat-inactivated at 60°C for 10 minutes. However, we recommend inactivating RNase R by adding EDTA (10 mM final concentration) to prevent unwanted RNA hydrolysis. Q: What are circular RNAs? A: Circular RNAs are single-stranded RNAs that form a covalently closed loop. These RNAs are resistant to exoribonucleases, such as RNase R (NEB #M0100), because there is no free end for these exoribonucleases to bind to. This resistance to exoribonucleases makes circular RNAs more stable than their linear counterparts. Q: Does RNase R (NEB #M0100) have star activity? A: Yes, adding a high concentration of RNase R to circular RNA samples can lead to circular RNA degradation. We recommend 1 unit of NEB RNase R per microgram of RNA (1:1 ratio) to prevent star activity. Q: Is it possible to digest RNase R (NEB #M0100)-resistant linear RNAs? A: Yes, we have tested two methods to digest RNase R-resistant linear RNAs: We recommend adding 1 unit of RNase R, 1 unit of XRN-1 (NEB #M0338), and 5 units of RppH (NEB #M0356) to 1 µg of RNA sample in 1X RNase R Reaction Buffer. The reaction should be incubated for 15 or 30 minutes at 37°C for efficient linear RNA removal. Pre-treat RNA samples with E. coli Poly(A) Polymerase (NEB #M0276) to lengthen the 3´ ends of the RNAs, which can improve RNase R binding and activity. The RNA needs to be cleaned up before proceeding with the RNase R reaction. Q: Can RNase R diluted in 1X Reaction Buffer be stored at 4°C? A: Yes. Functional testing confirms that RNase R diluted in 1X RNase R Reaction Buffer exhibits no significant loss of activity for up to one week when stored at 4°C.