Sonics VC40 40-watt (1988) - Manual (Page 14)

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If enzymes cannot be used, the following procedures should be considered: Freezing the
sample at -70
?
C overnight, then thawing it in water immediately prior to ultrasonic
processing.
Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue
cold and to process it quickly (within 30 minutes) after dissection. When working with
fresh tissue, the cells must be sonicated immediately at the time the GITC lysis solution is
added. This can be done by dispensing the lysing solution in the tube, adding the tissue
and immediately sonicating. Samples should never be left sitting in lysis solution,
undisrupted. La rge samples of hard tissues should be first treated in a blender or a
mechanical homogenizer.
Animal tissues that have been frozen after collection should be disrupted by grinding in
liquid nitrogen with a mortar and pestle. During this process, it is important that the
equipment and tissue remain at cryogenic temperatures. The tissue should be dry and
powdery after grinding. Grinding should be followed by thorough sonication in a GITC
lysis buffer. Processing frozen tissue in this way is cumbersome and time consuming, but
effective.
Cultured cells are normally easy to disrupt. Cells grown in suspension are collected by
centrifugation, rinsed with PBS to remove culture medium, and then lysed by sonicating
in a GITC lysis buffer. Placement of the vessel on ice while washing and lysing the cells
will further protect the RNA from endogenous RNases released during the disruption
process.
Soft, fresh plant tissue can often be disrupted by sonicating in a lysis buffer. Other plant
tissues, like pine needles, need to be ground dry, without liquid nitrogen. Some hard,
woody plant materials require freezing and grinding in liquid nitrogen prior to being
ultrasonically processed. Plant cell suspension cultures and calluses can typically be
sonicated in a lysis buffer within 2 minutes. The diversity of plants and plant tissue make
it impossible to give a single recommendation for all. However, most plant tissues
typically contain polysaccharides and polyphenols that can coprecipitate with RNA and
inhibit downstream assays. Treating a plant tissue lysate with polyvinylpyrrolidone
(PVP) will precipitate such problematic components from the lysate before the actual
RNA isolation is carried out.
Whenever possible, the tissues should be diced very small to permit movement within the
liquid. Tough tissues such as skin and muscle should be macerated first in a blender or
the like for about 10 seconds, and confined to a small vessel during ultrasonic treatment.
If sub-cellular particles are desired intact, the amplitude should be kept low, and the
processing time increased.
Yeast can be extremely difficult to disrupt because their cell walls may form capsules or
nearly indestructible spores. To process yeast, sonicate in a tube containing the sample,
guanidinium-based lysis buffer and small glass beads (0.5 – 1 mm). Pretreatment with