Bio-Rad Aurum™ Total RNA Mini Kit - Manual (Page 10)

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Elution Guidelines
• Apply elution solution directly to the membrane stack at the base of each RNA
binding column
Ribonucleases
• Although the components of this kit are provided free of contaminating
ribonucleases, great care must be taken not to contaminate the solutions
or the RNA binding columns. Gloves should always be worn when handling RNA
and should be changed frequently. Care should be taken to proceed through the
RNA isolation as quickly as possible
• Solutions that are prepared by the user (e.g., TE) should be treated with diethyl
pyrocarbonate (DEPC) to inactivate RNases. Add 1 ml DEPC per liter (final
concentration 0.1%) of solution to be treated, mix thoroughly,
and incubate the solution at 37°C for 1 hr. Autoclave the solution to
remove the DEPC
Note: DEPC is destroyed by primary amines (e.g., Tris). If a solution containing a
primary amine will be DEPC-treated, omit the amine in preparing the solution.
Perform the DEPC treatment as described above and add the amine to the
autoclaved solution once the solution has cooled
• Nondisposable, nonautoclavable plasticware should be rinsed with 0.1 M NaOH,
1 mM EDTA followed by several rinses with DEPC-treated water before use
• Glassware and other autoclavable items may be treated using the DEPC method
described above for nonautoclavable plasticware, or by baking for 4 hr at 300°C
• Work surfaces and micropipettors should be kept clean and wiped periodically
Disruption and Homogenization
Disruption methods facilitate lysis of the starting material at the beginning
of the RNA purification. Isolating RNA from nonadherent and adherent mammalian
cultures and from unicellular organisms typically involves a straight forward
disruption method, such as repeated pipetting up and down. However, for animal
and plant tissue, more vigorous disruption methods may be required in order to
expose cells in the interior of the tissue sample to the lysis buffer. Grinding tissue
with a mortar and pestle under liquid nitrogen greatly increases the cell surface
area exposed to the lysis buffer while simultaneously inhibiting ribonucleases.
Following lysis, the lysate often becomes very viscous due to the release of
genomic DNA into the solution. It is very important to reduce the viscosity of the
lysate using a homogenization method, as a viscous, heterogeneous solution may
cause the RNA binding column to clog. See Table 2 for a list of disruption and
homogenization methods recommended for a particular starting material.
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