RNA Purification

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. 

The disruption and homogenization methods are described in more detail below.

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.

Rotor–stator homogenizers thoroughly disrupt and simultaneously homogenize, in the presence of lysis buffer, animal tissues in 5–90 seconds depending on the toughness of the sample. Rotor–stator homogenizers can also be used to homogenize cell lysates. The rotor turns at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing. Foaming of the sample should be kept to a minimum by using properly sized vessels, by keeping the tip of the homogenizer submerged, and by holding the immersed tip to one side of the tube. Rotor–stator homogenizers are available in different sizes and operate with differently sized probes. Probes with diameters of 5 mm and 7 mm are suitable for volumes up to 300 µl and can be used for homogenization in microfuge tubes. Probes with a diameter of 10 mm or above require larger tubes.

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.

Cell and tissue lysates can be homogenized using a syringe and needle. High-molecular-weight DNA can be sheared by passing the lysate through a 20-gauge (0.9 mm) needle, attached to a sterile plastic syringe, at least 5–10 times or until a homogeneous lysate is achieved. Increasing the volume of lysis buffer may be required to facilitate handling and minimize sample loss.
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