How to use ultrasound for cell disruption and DNA/RNA extraction？

How to use ultrasound for cell disruption and DNA/RNA extraction？

Ultrasound is an efficient and commonly used technology in cell disruption and nucleic acid (DNA/RNA) extraction. Its core principle is to use cavitation effect and mechanical vibration to destroy cell structure and release internal substances. The following is an explanation from the specific mechanism, application process and key precautions:

I. Principle and process of ultrasound for cell disruption
The core of cell disruption is to destroy the cell membrane (animal cells) or cell wall + cell membrane (plants, bacteria, fungi, etc.) to release intracellular substances (such as nucleic acids, proteins, organelles, etc.). Ultrasound achieves this process through the following mechanisms:
1. Core principle: cavitation effect
Ultrasound is a high-frequency mechanical wave with a frequency higher than 20kHz. When the ultrasonic probe (amplifier of ultrasonic disruptor) is inserted into a liquid sample containing cells, high-frequency vibration (usually 15-50kHz) is generated, which triggers cavitation in the liquid medium:

High-frequency vibration causes a large number of tiny bubbles (cavitation bubbles) to form in the liquid. These bubbles expand and compress rapidly under pressure changes, and finally burst violently (collapse).
When bubbles burst, huge instantaneous energy (local high temperature, high pressure and strong shock waves) will be released, and the impact force generated will directly tear the membrane structure of the cell (cell membrane, cell wall) to achieve cell fragmentation.
2. Auxiliary effect: mechanical vibration and shear force
The high-frequency mechanical vibration of ultrasound will also directly generate shear force and friction force on the cells, further assisting in destroying the cell structure, especially for cells with thick cell walls (such as fungi and plant cells).
3. Fragmentation process (taking experimental operation as an example)
Put the cell suspension to be treated (such as bacterial culture fluid, tissue homogenate, etc.) into a centrifuge tube or beaker, insert the ultrasonic probe (horn), and the probe needs to be immersed in the liquid but not in contact with the container wall.

Turn on the ultrasonic instrument and set parameters (power, time, etc.): high-frequency vibration generates cavitation bubbles in the liquid, and the impact force generated by the bursting of bubbles directly destroys the cell structure and releases intracellular substances (including nucleic acids, proteins, metabolites, etc.).
2. Specific application of ultrasound in DNA/RNA extraction
The core steps of DNA/RNA extraction include: cell disruption to release nucleic acids → removal of impurities (proteins, lipids, polysaccharides, etc.) → purification of nucleic acids. The role of ultrasound is mainly concentrated in the first step - efficient cell disruption and nucleic acid release. The specific application scenarios and advantages are as follows:
1. Suitable sample types
Ultrasound is suitable for nucleic acid extraction of a variety of samples, including:

Animal tissue (such as liver, muscle): need to be cut into small pieces first, ultrasound can quickly disrupt cells and release nucleic acids;
Bacteria/fungi: cell walls are relatively hard, traditional methods (such as lysozyme treatment) are inefficient, and ultrasound can destroy cell walls through strong cavitation effect;
Cultured cells (adherent or suspended cells): direct suspension and then ultrasound, no complex pretreatment is required;
Plant tissue: contains cellulose and pectin, ultrasound can assist in breaking cell walls and releasing cell contents.
2. Key parameters in operation (affecting nucleic acid quality)
Ultrasonic treatment requires strict control of parameters to avoid nucleic acid degradation or excessive fragmentation (affecting subsequent experiments, such as PCR, sequencing, etc.):

Power: usually 50-300W (adjusted according to sample type, such as bacteria require higher power). Too high power will cause nucleic acid shearing to be too short, and too low power will result in insufficient fragmentation.
Working/interval time: Use "pulse" treatment (such as working 30 seconds + intermission 30 seconds) to avoid continuous ultrasonic heat generation leading to nucleic acid denaturation (DNA/RNA is easily degraded at high temperature).
Total treatment time: Depends on the sample (such as 1-3 minutes for animal cells and 3-5 minutes for bacteria). Excessive treatment will aggravate nucleic acid fragmentation.
Temperature control: Ice bath throughout the process (samples are placed on ice), because the ultrasonic process will generate heat (cavitation effect accompanied by local high temperature), low temperature can inhibit nuclease activity and protect nucleic acid stability.

3. Advantages and precautions
Advantages
High efficiency: Faster than traditional methods (such as grinding, repeated freezing and thawing, enzymatic hydrolysis) (usually completed within a few minutes), and more thorough disruption;
Universality: Applicable to a variety of sample types (animals, plants, microorganisms, etc.);
Easy to operate: No complex reagents are required, only ultrasonic instruments and basic centrifugation equipment are required.
Precautions
Avoid bubbles: If there are a large number of bubbles in the sample, the efficiency of the cavitation effect will be reduced, and the probe may overheat. Make sure that the probe is completely immersed in the liquid (the liquid level is about 1-2cm);
Nuclease inhibition: After disruption, lysis solution (including EDTA, detergent, etc.) should be added in time to inhibit intracellular nucleases (such as RNase that easily degrades RNA);
Sample volume: The single processing volume should not be too large (usually ≤5mL), otherwise the ultrasonic energy distribution will be uneven and the disruption effect will vary greatly.

Summary
Ultrasound efficiently disrupts cells through cavitation effect and mechanical vibration, providing a key "release step" for DNA/RNA extraction. The core is to optimize power, time and temperature to fully disrupt cells while maximizing the protection of nucleic acid integrity. This technology is widely used in the pre-processing stage of molecular biology experiments (such as gene cloning, qPCR, transcriptome analysis, etc.) and is an important tool for nucleic acid extraction.