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RNeasy Mini Handbook 10/2019
53
Protocol: Purification of Total RNA from Yeast
This protocol requires the RNeasy Mini Kit.
Disrupting yeast cells
This protocol for purifying total RNA from yeast provides 2 alternative methods of disrupting
the walls of yeast cells:
Enzymatic lysis: This method requires digestion of the cell wall with zymolase or lyticase
to convert cells to spheroplasts. 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.
Mechanical disruption: This method uses high-speed agitation in the TissueLyser or other
bead mill in the presence of glass beads and Buffer RLT to lyse yeast cells and release
RNA.
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.
Determining the correct amount of starting material
It is essential to use the correct amount of starting material in order to obtain optimal RNA
yield and purity. The maximum amount depends on:
The RNA binding capacity of the RNeasy spin column (100 µg RNA)
The volume of Buffer RLT required for efficient lysis (the maximum volume of Buffer RLT
that can be used limits the maximum amount of starting material to 5 x 10
7
yeast cells)
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