Homogenization of Soft Animal Tissue with a Rotor Stator
Certain animal tissues are relatively easy to homogenize and can be processed by a variety of methods. Liver, thymus, fat, and brain, all soft tissues, can be homogenized by bead beating, shearing with rotor stators, and by cryogenic grinding. Softer tissues have less connective tissue than muscle and thus much easier to homogenize.
The major considerations in deciding on a homogenization method are sample size and target analyte. Rotor stators are useful for a limited number of samples while bead beating is the best high throughput method. For RNA extractions where RNAlater is NOT used, grinding frozen samples can be performed with the CryoGrinder or in vials using a Geno/Grinder.
Pourrut, Xavier, Marc Souris, Jonathan S Towner, Pierre E Rollin, Stuart T Nichol, Jean-Paul Gonzalez and Eric Leroy. 2009. Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus. BMC Infectious Diseases 2009, 9:159
Homogenizing with Rotor Stator Homogenizer
Commonly referred to as handheld homogenizers, rotor stator homogenizers are effective for soft tissues and small pieces of resilient tissues, such as muscle. Samples with significant amounts of connective tissue or tendon are more difficult to homogenize as the elastic tissues can get caught up in the shaft of the rotor stator. This is less of a problem with softer tissues.
The rotor stator needs to be matched with the sample volume. Product literature suggests that samples as small as 30 µl can be homogenized, but in practice that volume isn't practical. Generally samples of 200 µl up to many liters can be processed, but the correct attachment is needed. Assuming the correct rotor stator is available, place the sample in a tube or beaker that is sufficiently narrow so that the slots on the bottom of the rotor stator are easily submersed.
If protein degradation is an issue, the sample container can be placed on ice turning the processing. The shaft will generate heat, thus cooling the sample can slow down unwanted reactions.
Turn the homogenizer on low and slowly work the rotor stator into the sample. Avoid raising the rotor stator out of the buffer as air will be whipped into the homogenate. Air and frothing can be detrimental to many proteins.
The speed can be slowly increased so that the homogenate circulates through the tip of the rotor stator. Harder, more resilient tissues will need more processing to breakdown fibrils.
Homogenization can be optimized by measuring the release of cellular enzymes. We commonly use lactate dehydrogenase to measure homogenization efficiency. A time course assay can be performed on the homogenate and assayed for enzyme release. For more on the LDH assay see this link.