Sonics VC750 (Serial No. "Y through "AB")" - Manual (Page 24)

21
the sample remains trapped in intact cells and, therefore, is unavailable for
subsequent purification. For most samples, thorough disruption can be monitored by
close inspection of lysate after disruption. There should be no visible particulates,
except when disrupting materials containing hard, non-cellular components, such as
connective tissue or bone.
When processing difficult cells, pretreatment with an enzyme to “weaken” the cell
walls is beneficial. Lysozyme, byaluronidase, glycosidase, glucalase, lyticase,
zymolase and lysostaphin digestion are among the enzymatic methods frequently
used with yeast. Lysozyme, zymolase and lysostaphin digestion are among the
enzymatic methods frequently used with bacteria and yeast to dissolve a coat,
capsule, capsid or other structure not easily sheared by ultrasonics. Enzymatic
treatment is usually followed by sonication in a GITC lysis buffer. Collogenase may be
used with collogen, lysostaphin with staphylococcus, and trypsin hyaluronidase with
liver and kidney.
Yeast can be extremely difficult to disrupt. To process yeast sonicate in a tube
containing the sample, guanidinium-based lysis buffer and small glass beads (0.5 – 1
mm). Enzymatic pretreatment as outlined above is strongly recommended.
To disrupt filamentous fungi, scrape the mycelial mat into a cold mortar, add liquid
nitrogen, grind to a fine powder with a pestle, then sonicate in lysis buffer to solubilize
completely. As fungi may also be rich in polysaccharides, pretreatment with
polyvinylpyrrolidone (PVP) may be beneficial.
Disruption of cells found in soil and sediments is accomplished by 1) isolating the
bacterial cells from the material prior to the RNA isolation procedure. This is
accomplished by homogenization of wet soil in a mechanical blender followed by a
slow speed to pellet the bacteria cells. From this point, cells can be lysed as
described above for bacteria, or 2) isolating RNA from the soil or sediment directly.
For example, adding soil to a diatomaceous earth and lysis buffer, and then
sonicating. The sample is then centrifuged to remove solid debris.
Cultured cells are relatively easy to disrupt. Cells grown in suspension are collected
by centrifugation, rinsed to remove culture medium, and then lysed by sonicating in
GITC lysis buffer. Placing the flask or plate on ice while washing and lysing the cells
will further protect the RNA from endogenous Rnases released during the disruption
process.