Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 13)

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• Nondisposable, nonautoclavable plasticware should be rinsed with 0.1 M
NaOH, 1 mM EDTA followed by several rinses with DEPC-treated water before
use
• Glassware and other autoclavable items may be treated using the DEPC
method described above for nonautoclavable plasticware, or by baking for 4
hr at 300°C
• Work surfaces and micropipets should be kept clean and wiped periodically
Sample Disruption and Homogenization
Successful isolation of total RNA is dependent on the efficient disruption and
homogenization of cells and tissues. Cell and tissue disruption is the physical
breakdown of cell walls and plasma membranes, usually done using mechanical
or enzymatic techniques. Efficient disruption facilitates the lysis of the starting
material and release of all the RNA contained in the sample, ensuring a high yield
of RNA. Incomplete disruption results in column clogging and significantly
reduced RNA yields. After disruption, proper mixing of the lysate is necessary to
produce a homogenous solution for efficient passage of the lysate through the
Aurum RNA binding mini column and for RNA to properly bind to the silica
membrane.
Isolation of RNA from nonadherent and adherent mammalian cultures typically
involves a straightforward disruption method such as repeated pipetting up and
down or passing through an 18-gauge needle and syringe. For animal and plant
tissues, more vigorous disruption methods may be required to increase the cell
surface area exposed to the PureZOL RNA isolation reagent while simultaneously
inhibiting RNases. Tissue disruption can be performed by first grinding with a
mortar and pestle under liquid nitrogen and then using either a rotor-stator
homogenizer or a bead mill homogenizer (see manufacturer instructions for more
detail). If a homogenizer is not available, passing the tissue sample through an
18-gauge needle and syringe may also work. However, RNA yields will not be as
high as when using a homogenizer.
Bacteria and yeast cells have thick cells walls that are difficult to break. Repeated
pipetting up and down or passing the sample through an 18-gauge needle and
syringe may not be sufficient for lysing bacteria and yeast cells. More vigorous
physical disruption methods, such as using a rotor-stator homogenizer or a bead
mill homogenizer (see manufacturer instructions for more detail), may be required
in order to lyse the bacterial and yeast cells.
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