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22
RNeasy Mini Handbook 10/2019
Animal and yeast cells can be pelleted and then stored at −90 to −65°C until required for RNA
purification. However, if performing RNA purification from yeast cells with enzymatic lysis,
only freshly harvested samples can be used.
Disrupting and homogenizing starting material
Efficient disruption and homogenization of the starting material is an absolute requirement for
all total RNA purification procedures. Disruption and homogenization are 2 distinct steps:
Disruption: Complete disruption of cell walls and plasma membranes of cells and
organelles is absolutely required to release all the RNA contained in the sample.
Different samples require different methods to achieve complete disruption. Incomplete
disruption results in significantly reduced RNA yields.
Homogenization: Homogenization is necessary to reduce the viscosity of the lysates
produced by disruption. Homogenization shears high-molecular-weight genomic DNA
and other high-molecular-weight cellular components to create a homogeneous lysate.
Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin
column membrane and therefore significantly reduced RNA yields.
Some disruption methods simultaneously homogenize the sample, while others require an
additional homogenization step. Table 3 (page 23) gives an overview of different disruption
and homogenization methods, and is followed by a detailed description of each method. This
information can be used as a guide to choose the appropriate methods for your starting
material.
Note: After storage in RNAprotect Tissue Reagent, tissues become slightly harder than fresh or
thawed tissues. Disruption and homogenization of these tissues, however, is usually not a
problem.
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