Rapid, high-throughput homogenization of embryonic or larval zebrafish (Danio rerio)
Introduction
Danio rerio are an important model system, ideal for use in many developmental, cellular, and molecular biology experiments. As use of this highly tractable model organism increases, so do the needs for more effective and efficient methods regarding their use. Homogenization is commonly used for extraction of proteins, nucleic acids, and chemical analytes from tissue or whole organisms. Here we describe a protocol for highly consistent, high-throughput homogenization of whole or dissected larval or embryonic zebrafish. Twenty-four D. rerio samples may be homogenized in a total time of five minutes.
Materials
Reagents
Lysis / Extraction Buffer (as appropriate for your downstream application)
Equipment
Bullet BlenderTM
Zirconium Oxide Grinding Beads (0.5 mm or 1.0 mm)
Procedure
1. Place 10-300mg of zebrafish into microcentrifuge tubes. Unhatched fish do not require prior dechorionation.
2. To each tube, add two masses of zirconium oxide grinding beads for each mass of sample.
3. Add 2 volumes of buffer for every mass of zebrafish (for example, with 100mg of fish use 200ml of buffer). You may choose your buffer as appropriate for your downstream application.
4. Close the microcentrifuge tubes and place the tubes into the Bullet BlenderTM (they do not need to be balanced).
5. Set controls for SPEED 8 and TIME 3 minutes, then start the run.
6. Remove tubes and proceed with your downstream application.
Troubleshooting
If your homogenization is not satisfactory, be sure you have the correct ratio of sample to beads to buffer. If you have the correct ratio and still are not achieving satisfactory homogenization, run your samples for another three minutes at speed 10.
Critical Steps
All of the steps are critical. The homogenization will be incomplete if you miss a step.
Anticipated Results
The Bullet BlenderTM, when used appropriately and in conjunction with an appropriate cell lysis buffer, will achieve a high degree of homogenization while maintaining genomic integrity and RNA integrity. RNA integrity numbers of over 8.0 may be expected.
References
Acknowledgements
Keywords
homogenization, zebrafish, embryonic zebrafish, larval zebrafish, zebrafish homogenization, homogenize zebrafish, homogenize, lyse, lysis, disruption, extraction, Danio rerio, D. rerio
Homogenization of 2-6 days post-fertilization (dpf) zebrafish.
Before / after images of zebrafish homogenizaton in the Bullet BlenderTM. Note that the homogenate will be darker at later ages due to increasing melanization of the zebrafish with age.
