Figure 1. Quantitative Hierarchy of RNase Activity in Mouse Tissues. Mouse tissues were dounced in non-denaturing, neutral pH buffer (50 mM NaCl and 1 mM Mg2+), and total RNase activity was measured using Ambions fluorescence-based RNaseAlert QC System. Lysates were treated with EDTA (5 mM) or Ambions RNase Inhibitor protein (40 U/100 l reaction).
The tissues profiled for intrinsic RNase activity belonged in one of two groups. While RNases in Group A tissues were insensitiveto EDTA (and thus, Mg2+ independent), these tissues contained very high levels of RNase activity that were effectively inhibited by Ambions recombinant RNase Inhibitor, an inhibitor of RNase A-type enzymes. Group B tissues exhibited much lower levels of RNase activity that were more thoroughly inhibited by EDTA than by Ambions RNase Inhibitor (both before and after heating the lysates to inactivate endogenous inhibitors). Because RNase A enzyme activity is unaffected by the addition of EDTA, whereas other RNases are usually EDTA-sensitive, these results suggest that the RNase A family of enzymes are the source of RNase activity in Group A tissues, whereas a more functionally heterogeneous group of RNases are active in Group B tissues.
For RNA isolation from tissues containing very high levels of RNase, we recommend TRI Reagent (see RNA, DNA, and Protein from a Single Sample and High Throughput RNA Isolation for High Performance Microarray Analysis). For tissues exhibiting more typical levels of RNase activity, Ambions convenient hands-free MELT Total RNA Isolation System maximizes yields with minimal handling (see Watch Your Tissue MELT into High Quality Total RNA).
Julie Krosting, Gary Latham Ambion, Inc.
Cat# Product Name Size 1839 MagMAX-96 for Microarrays Total RNA Isolation Kit 96 purifications 1964 RNaseAlert Lab Test Kit 25 rxns 1966 RNaseAlert QC System 5 x 96 assays 1982 MELT Total Nucleic Acid Isolation System 50 purifications 2682 RNase Inhibitor (Cloned) 40 U/l 2500 U 2684 RNase Inhibitor (Cloned) 40 U/l 10,000 U 9738 TRI Reagent 100 ml