Loading ...
Loading ...
Loading ...
RNeasy Mini Handbook 10/2019
13
Purification of Total RNA from Animal Tissues
Fresh, frozen, or RNAprotect stabilized tissue (up to 30 mg, depending on the tissue type) is
disrupted in Buffer RLT and homogenized. An overview of disruption and homogenization
methods is given on page 22. Ethanol is then added to the lysate, creating conditions that
promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the
RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently
washed away, and high-quality RNA is eluted in RNAse-free water.
Purification of Total RNA from Yeast
This protocol is for the purification of total RNA from up to 5 x 10
7
yeast cells. Two alternative
methods of disrupting yeast cell walls are provided: enzymatic lysis or mechanical disruption.
In general, both methods function equally well. For some applications, enzymatic lysis might
be preferable since no additional laboratory equipment is required. Mechanical disruption,
however, is well-suited for time-course experiments where enzymatic digestion incubations are
not practical.
The enzymatic lysis method uses zymolase or lyticase digestion of the cell walls to convert cells
to spheroplasts, which are then used in the RNeasy procedure. For samples of up to 5 x 10
7
yeast cells, spheroplasts are separated from the digestion mixture by centrifugation before
being lysed. For samples of up to 2 x 10
7
yeast cells, the digestion mixture is used directly in
the RNeasy procedure without prior separation of the spheroplasts. After addition of Buffer
RLT and ethanol, samples are loaded onto the RNeasy Mini spin column. Total RNA binds to
the RNeasy membrane, contaminants are efficiently washed away, and high-quality RNA is
eluted in RNase-free water.
Loading ...
Loading ...
Loading ...