High quality and high yield RNA extraction is of paramount importance for gene expression profiling and other associated analytical techniques. The first step in this process is the disruption of the tissue into small particle sizes. A number of technologies exist for this however; they have changed little in a number of years and suffer from a number of drawbacks that make them less than desirable. Often, these techniques require direct contact with the sample, which given the extreme sensitivity of the analytical processes, creates extensive decontamination to eliminate sample cross-talk.

An example of RNA yield and quality of RNA extraction can be seen in the Figure below:



The Covaris AFA process is ideally suited to sample disruption (homogenization). Conversely, in a rotor/stator process (e.g., Polytron), shearing is limited to a particle size which is defined by the distance between the blades. Bead beaters, and mortar and pestle also have shear-stress size limits. For example, a mortar and pestle grinding in liquid nitrogen produces a particle of ~100um. At this size, the particle "powder" is not a molecule, hence strong chemicals are required to diffuse into the sample to complete the standard inefficient process.

Feature	Benefit
Controllable mechanical energy	Highest yield of RNA Fast processing of sample – higher quality RNA Disrupts all tissue types in conjunction with CryoPrep™ Ability to completely homogenize samples (<10µm particle size)
PC controlled	Highly reproducible
Non-contact	No sample contamination concerns No instrument clean up post processing Safe – no aerosol risk
Automated – E series	Walk away sample disruption
Isothermal	Disruption temperature controllable (4 – 40°C)

How do you use the system?

If a tissue is hard or fibrous, then an initial cryopulverization method may be required. Use of the Covaris CryoPrep™ and TissueTube system greatly facilitates this step as it both fractures extracellular matrices (e.g., connective tissue) and increases the surface area for the acoustic extraction (more details here).

RNA extraction buffer (e.g., a guanidinium isothiocyanate buffer) is added to a frozen tissue sample specimen with or without a CryoPrep pretreatment in a TissueTube. A lid is placed on the tube and the tube placed into the Covaris instrument. The instrument is then activated and the homogenization started. Typical homogenization times are 30 to 60 seconds. A proven combination is using:

1. The TissueTube for collection and rapid, thermal stabilization;
2. The CryoPrep for rapid pulverization; and
3. The S2 unit for rapid acoustic disruption and chemical stabilization.

This three step process is also beneficial for the isolation of labile biomolecules (e.g., active proteosome complexes). Often the "extraction" buffer is the stabilization buffer for the target biomolecule.