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 Ambion’s fluorescence-based RNaseAlert® QC System. Lysates were treated with EDTA (5 mM) or Ambion’s RNase Inhibitor protein (40 U/100 µl 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 Ambion’s 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 Ambion’s 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). For tissues exhibiting more typical levels of RNase activity, Ambion’s 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.