Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 20)

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 cell 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.
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.
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