RNA isolation: Disruption and homogenization of starting materials
Efficient disruption and homogenization of the starting material is an absolute requirement for all total RNA isolation procedures. Disruption and homogenization are two distinct steps.
- Disruption: Complete disruption of tissue structure, cell walls, and plasma membranes of cells is absolutely required to release all the RNA contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced yields.
- Homogenization: Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption. Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA and therefore significantly reduced yields.
Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step. The infographic gives an overview of different disruption and homogenization methods suitable for various starting materials. It can be used as a guide to choose the appropriate method for the starting material with which you are working.
Disruption and homogenization using bead mills
In disruption using a bead mill, the sample is agitated at high speed in the presence of beads. Disruption and simultaneous homogenization occur by the hydrodynamic shearing and crushing action of the beads as they collide with the cells. Disruption efficiency is influenced by:
- Size and composition of beads
- Ratio of buffer to beads
- Amount of starting material
- Speed and configuration of agitator
- Disintegration time
The optimal beads to use are 0.1 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, and 3–7 mm stainless steel beads for animal and plant tissues. It is essential that glass beads are pretreated by washing in concentrated nitric acid. Alternatively, use commercially available acid-washed glass beads. All other disruption parameters must be determined empirically for each application. Plant material as well as the beads and disruption vessels can be precooled in liquid nitrogen, and disruption should be performed without lysis buffer. Dry, cryogenic grinding is also used for animal tissue. Cryogenic grinding (regardless of whether in a bead mill or by mortar and pestle) does not homogenize the sample, unlike when lysis buffer is used.
Disruption and homogenization using rotor–stator homogenizers
Disruption using a mortar and pestle
For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen-cooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the procedure.
Note: Grinding the sample using a mortar and pestle will disrupt the sample, but it will not homogenize it. Homogenization must be performed separately before proceeding.