Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 26)

Note: Do not wash cells prior to the addition of PureZOL as this could
increase the possibility of mRNA degradation.
3. Once the sample has been disrupted in PureZOL, incubate the
lysate at room temperature for 5 min to allow the complete
dissociation of nucleoprotein complexes.
Note: Following the disruption step, the sample can be stored at –70°C for
at least one 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 min to completely dissolve salts. Avoid extended treatment at
37°C, which can cause chemical degradation of the RNA.
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 min at 4°C following the 5 min 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 4.
For lipid-rich samples, avoid transferring the excess fat that collects as a
top layer. Carryover of the solid debris can cause column clogging and
affect RNA sample purity.
4. Add 0.2 ml of chloroform to the lysate, then cover and shake
vigorously for 15 sec. Do not vortex!
5. Incubate for 5 min at room temperature while periodically
mixing the sample.
6. Centrifuge at 12,000 x g for 15 min at 4°C.
Following centrifugation, the mixture will separate into three phases: an
upper, colorless aqueous phase, a white interphase, and a lower, red
organic phase. RNA will be exclusively in the aqueous phase, while DNA
and proteins remain in the interphase and organic phase. The volume of
the aqueous phase should be approximately 600 µl, or 60% of the volume
of PureZOL used in the initial disruption.
If removal of contaminating DNA is a requirement, prepare DNase I enzyme by
following steps a–b below while centrifuging the samples for phase separation:
a. DNase I is provided as a lyophilized powder. If the DNase has already
been reconstituted, skip to step b. Otherwise, reconstitute the DNase I
by adding 250 µl of 10 mM Tris, pH 7.5 (not provided) to the vial and
mix by pipetting up and down briefly. Do not vortex! See Section 4,
Materials and Equipment Required (Not Provided in the Kit), on how to
prepare 10 mM Tris, pH 7.5.
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