Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 25)

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up to 100 mg of freshly dissected tissue into a 2.0 ml microcentrifuge tube
and add 1 ml of PureZOL. Disrupt the sample for 30–60 sec using a
rotor-stator homogenizer or a bead mill homogenizer (refer to manufacturer
instructions for more details). Although not as effective, passing the tissue
sample through an 18-gauge needle and syringe can be used for sample
disruption if a homogenizer is not available. Pass the sample through the
needle and syringe until no more solid tissue is left in the lysate. The
sample volume should not exceed 10% of the volume of PureZOL used for
disruption. Proceed to step 3.
Frozen Tissue
Grind up the frozen tissue to fine powder with a mortar and pestle under
liquid nitrogen. Avoid thawing the sample by periodically adding liquid
nitrogen to the mortar. Weigh up to 100 mg of tissue and transfer the
sample into a 2.0 ml microcentrifuge tube. Add 1 ml of PureZOL and
disrupt for 30–60 sec using a rotor-stator homogenizer or a bead mill
homogenizer (refer to manufacturer instructions for more details). Although
not as effective, passing the tissue sample through an 18-gauge needle
and syringe can be used for sample disruption if a homogenizer is not
available. Pass the sample through the needle and syringe until no more
solid tissue is left in the lysate. The sample volume should not exceed 10%
of the volume of PureZOL used for disruption. Proceed to step 3.
Cells Grown in a Monolayer
Cells grown in a monolayer should be lysed with PureZOL directly in the
culture dish. Aspirate the culture medium and immediately add 1 ml of
PureZOL to a 10 cm
2 dish. Pass the lysate through a pipet several times.
The amount of PureZOL added is dependent on the area of culture dish
(1 ml per 10 cm
2 ) and not on cell number. Insufficient volumes of PureZOL
may result in DNA contamination. Proceed to step 3.
Note: Do not wash cells prior to the addition of PureZOL as this could
increase the possibility of mRNA degradation.
Suspension Cells (Mammalian, Plant, Bacterial, or Yeast)
Pellet the cells by centrifuging at 3,000–5,000 x g for 2 min. Immediately
lyse by adding 1 ml of PureZOL to 1 x 10
7 cultured mammalian and plant
cells, 2.4 x 10 9 of gram-positive or gram-negative bacteria, or 3.0 x 10 7 of
yeast (equivalent to 3 OD•ml of yeast). Pass the lysate through a pipet or
an 18-gauge needle and syringe several times. To improve the efficiency of
the cells lysis process, a rotor-stator homogenizer or a bead mill
homogenizer is recommended to disrupt the cell walls of yeast and
bacteria. Bacteria and yeast lysate can also be heated to 55°C for 10 min
prior to adding chloroform to increase lysis effectivity of PureZOL. Proceed
to step 3.
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