Workdays can sometimes seem very long when you have to use mortar and pestle in the lab to disrupt your samples.
And sample disruption can be particularly tedious, time-consuming and prone to cross-contamination when having to process a number of different samples at the same time.
Although mortar and pestle are still being used today in the lab for grinding solid samples or frozen tissue with liquid nitrogen, it’s good to be aware of the variety of alternative methods, tools and reagents available that can also help disrupt your samples effectively to isolate RNA, DNA or protein.
In order to develop a suitable method that disrupts your samples according to your needs, it’s important to know:
1. How to best collect and harvest your sample
2. The characteristics of your target molecule
3. The sample throughput that is needed
This information can help you decide which disruption or homogenization method – chemical, mechanical or a combination of the two methods – will most effectively destroy your sample and obtain your final homogenate.
Chemical disruption methods
Agents you can use for chemical disruption may include:
• Surfactants (such as detergents)
Surfactants will disrupt the interface between hydrophilic and hydrophobic systems. They may be anionic, cationic or nonionic, depending on their electrical charge.
Table 1. Examples of detergents used for sample preparation
|SDS||Anionic||Disrupts membranes and denatures proteins.|
|CTAB||Cationic||Isolates DNA from plants. Polysaccharides associated with plants are insoluble in CTAB and high concentrations of NaCl. Can be used to effectively separate DNA from plant carbohydrates.|
|Triton X-100||Nonionic||Lyses cells to extract protein or organelles, or to permeabilize the membranes of living cells.|
|Tween 20||Nonionic||Lyses mammalian cells, saturates binding sites on surfaces. Can be used in foods, pharmaceuticals, and in wash solutions for assays.
Chaotropes, however, disrupt the hydrogen bonding network between water molecules, and are widely used for nucleic acid isolation procedures that use silica-based resins or gels for purification.
Enzymes such as proteases, cellulases or Proteinase K can be used for treatment of tissues, plants and cells. Enzymes are very effective for processing cell walls or extracellular matrices to obtain your nucleic acid of interest.
Mechanical disruption methods
Mechanical disruption methods may include ultrasonication, grinding, beating or shearing.
Ultrasonication is the most effective homogenization method for cell suspensions and microorganisms. By using pressure, it disrupts cell membranes in seconds and releases cellular content. For solid tissue, however, you should consider a different method, as ultrasonication generates an enormous amount of heat – which can denature many proteins.
For simple and rapid homogenization of cell and tissue lysates, you can consider using a biopolymer-shredding system such as the QIAshredder. Designed in a microcentrifuge spin-column format, the QIAshredder homogenizes cell and tissue lysates to reduce viscosity in a chemically inert manner – thereby avoiding the binding of nucleic acids.
Homogenization shears the high molecular weight genomic DNA and other high molecular weight cellular components to create a homogenous lysate. It’s important to note that homogenization with the QIAshredder cannot replace either mechanical tissue disruption or enzymatic cell wall lysis – two processes that are fundamental for successful RNA isolation from all sample types. Once complete disruption has been accomplished, the QIAshredder homogenizer can be used in place of needle and syringe or in place of rotor-stator homogenization.
Rotor-stators, such as the TissueRuptor, consist of an outer stationary tube and an inner-turning shaft connected to a motor. The blade of the probe rotates at a high speed, causing simultaneous disruption and homogenization of individual samples through a combination of turbulence and mechanical shearing. You can use it for a wide range of samples including human, animal and plant tissues.
Using transparent disposable probes allows you to visually monitor sample disruption. It also minimizes cross-contamination and time spent cleaning the probe. Alternatively, you may also use steel probes, which can be decontaminated and cleaned through autoclaving.
To process more samples at a time, various bead mills are available on the market such as the TissueLyser LT or the TissueLyser II. Different adapter sets are available to process up to 192 samples at a time through high-speed shaking, beating and grinding. Sample disruption may typically use one or two beads per sample, and a grinding ball may be used for hard-to-disrupt samples. Accessories such as bead dispensers can help increase work efficiency. Some suppliers may offer tubes that are pre-filled with several ceramic beads. This convenience, however, also comes with an additional cost. Table 2 shows which beads and bead size you can use for a given starting material.
Table 2. Recommended bead type and size based on starting material
|Starting material||For disruption of||Recommended bead type||Recommended bead size (mean diameter)|
|Up to 30 mg fresh or frozen tissue||Bacteria||Glass beads||0.1–0.6 mm|
|Yeast / unicellular animal cells||Glass beads||0.5 mm|
|Human, animal and plant tissue||Stainless steel beads
Tungsten carbide beads
For tough tissue samples, use one or two 7-mm beads to improve disruption efficiency.
|4–10 ml per jar; for fresh, frozen or lyophilized tissues||Hard samples like bone or plants||Stainless steel grinding balls
Teflon grinding balls
When using glass beads, it is also essential that the beads be pretreated before use by washing in concentrated nitric acid. Pretreated (acid-washed) beads can be purchased from many vendors of biological supplies. Be aware that phenol/guanidine-based lysis reagent buffers – often used for RNA extraction procedures – react with Tungsten Carbide beads and should not be used.
We have gathered 3 key resources for you to download and gain additional insights on effective sample disruption and homogenization:
1. An Application Note on effective mechanical sample lysis for reliable pathogen identification
2. A one-page guide for completing disruption and homogenization of all starting materials
3. A Product Profile on TissueRuptor and TissueLyser disruption systems
Download all 3 resources for free!