( Protecting RNA During Isolation ...)RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control

Protecting RNA During Isolation
RNase activity varies dramatically across different tissues. A comparison of total RNase activities for 8 different mouse tissues showed that total RNase activity spans a 181,000-fold range from pancreas to brain (Figure 1), which highlights the level of RNase control required for successful RNA isolation.

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 RNas
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