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24
RNeasy Mini Handbook 10/2019
For animal tissues, the optimal beads are 3–7 mm diameter stainless steel beads, and for yeast
cells, the optimal beads are 0.5 mm diameter glass beads. It is essential that glass beads are
prewashed in concentrated nitric acid. All other disruption parameters must be determined
empirically for each application. The protocol for RNA purification from yeast (page 53)
describes how to perform mechanical disruption of yeast cells with glass beads. For guidelines
on disruption and homogenization of animal tissues using the TissueLyser system, refer to the
TissueLyser Handbook
. For other bead mills, please refer to suppliers’ guidelines for further
details.
Plant tissues can be disrupted using the TissueLyser in combination with stainless steel or
tungsten carbide beads. In this case, plant material, beads, and disruption vessels must all be
precooled in liquid nitrogen, and disruption is performed without lysis buffer.
Disruption and homogenization using rotor–stator homogenizers
Rotor–stator homogenizers thoroughly disrupt and simultaneously homogenize, in the presence
of lysis buffer, single samples of animal tissues in 15–90 s depending on the toughness and
size of the sample. Rotor–stator homogenizers can also be used to homogenize cell lysates.
The rotor turns at a very high speed, causing the sample to be disrupted and homogenized by
a combination of turbulence and mechanical shearing. Foaming of the sample should be kept
to a minimum by using properly sized vessels, keeping the tip of the homogenizer submerged,
and holding the immersed tip to the side of the tube. Rotor–stator homogenizers are available
in different sizes and operate with differently sized probes. Probes with diameters of 5 mm
and 7 mm are suitable for volumes up to 300 µl and can be used for homogenization in
microcentrifuge tubes. Probes with a diameter of 10 mm or above require larger tubes. In
addition, round-bottomed tubes allow more efficient homogenization than conical-bottomed
tubes.
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