Bio-Rad PureZOL™ RNA Isolation Reagent - Manual (Page 11)

100 mg of tissue and transfer the sample into a suitable sized
tube for disruption and homogenization. Add 1 ml of PureZOL
reagent to every 50–100 mg of the frozen ground tissue and
immediately homogenize for 30–60 seconds using a rotor-stator
or bead mill homogenizer (refer to manufacturer's instructions for
details). Alternatively, take a small chunk of the frozen tissue
(equivalent to 50–100 mg) and drop it into 1 ml of PureZOL
reagent and immediately homogenize the sample. The sample
volume should not exceed 10% of the volume of PureZOL used
for homogenization.
2. Incubate the lysate at room temperature for 5 minutes once
the sample has been disrupted in PureZOL, to allow the
complete dissociation of nucleoprotein complexes.
Following the disruption step, the sample can be stored at
–70°C for at least 1 month. To process frozen lysates, samples
should be thawed at room temperature. If necessary, heat
samples to 37°C in a water bath for 5–10 minutes to completely
dissolve salts. Avoid extended treatment at 37°C since this can
cause chemical degradation of the RNA.
Note: It is recommended that lysate from tissues that are rich in
fat, polysaccharides, proteins, and extracellular material be
centrifuged at 12,000 x g for 10 minutes at 4°C following the
5 minute incubation at room temperature. This step removes
any solid insoluble debris that was left after the disruption step.
Transfer the supernatant into a new 2.0 ml microcentrifuge tube
without aspirating the pellet, then proceed to step 3. For lipid-
rich samples, avoid transferring the excess fat that collects as a
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