Ribonuclease R (RNase R)
Product DetailsShow Hide
Ribonuclease R includes both the enzyme and an optimised 10X Reaction Buffer.
Ribonuclease R (RNase R)
Key featuresShow Hide
- Specific: Digests all linear RNAs but does not digest lariat or circular RNA structures, or double stranded RNA with 3' overhangs shorter than seven nucleotides
- Heat Inactivated: Heat at 65 °C for 20 minutes to kill enzyme activity
- Valuable: Use the unique properties of this exoribonuclease to study alternative splicing and gene expression
Size: 250 units
Ribonuclease R (RNase R) from E. coli is a magnesium-dependent 3´ to ;5´ exoribonuclease that digests essentially all linear RNAs but does not digest lariat or circular RNA structures 1,2, or double stranded RNA with 3´ overhangs shorter than seven nucleotides. 2 Most cellular RNAs will be digested completely by RNase R, with the exception of tRNAs, 5S RNA, and intron lariats (Figure. 1). The 3´ tails of lariats will be trimmed by RNase R to the branch point nucleotide, where there is a 2´,5´-phosphodiester linkage. Lariats are produced during pre-mRNA splicing of intron regions and can be isolated from a mixture of total RNA by digestion with RNase R. The MasterPure™ RNA and Yeast RNA Purification Kits are ideal for such total RNA preparations.
- Alternative splicing and gene expression studies.
- Intron cDNA production.
- Intronic screening of cDNA libraries.
Lariat RNAs isolated using this method can be used as a template to produce labelled cDNA as a target for microarrays containing potential intron sequences, or for tiling arrays containing overlapping regions of complete chromosomes or genomes. The cDNA produced will not be a linear representation of the intron, but the sequences contained in it will be intron-derived.
Figure 1. Schematic overview showing processing of intron lariats by RNase R.
- Suzuki, H. et al. (2006) Nucleic Acids Res. 34, 63.
- Vincent, H.A. and Deutscher, M. P. (2006) J. Biol. Chem. 281, 29769.
Note: RNase R requires low (0.1-1.0 mM) magnesium concentrations for activity. Low EDTA concentrations in substrate RNA solutions can negatively affect RNase R activity. Additional MgCl 2 up to 1 mM final concentration can be used to compensate for EDTA in the substrate. Optimal activity is at 37 °C.
Unit Definition: One unit of RNase R converts 1 µg of poly(A) into acid-soluble nucleotides in 10 minutes at 37 °C under standard assay conditions.
Storage Buffer: RNase R is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.
RNase R 10X Reaction Buffer: 0.2 M Tris-HCl (pH 8.0), 1 M KCl, and 1 mM MgCl2.
Quality Control: RNase R is function-tested in a reaction containing a mixture of linear and circularized RNA oligonucleotides. Only the linear RNA is digested.