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Protecting RNA During Isolation
RNase Activity in
Mouse Tissue:
Classification, Hierarchy, and Methods for Control
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.
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| 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).
Scientific Contributors
Julie Krosting, Gary Latham • Ambion, Inc.
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