I’ve been trying to use a homogenization machine to simplify RNA isolation for microarrays, and break C. elegans seems to be more difficult that I thought. Those machines are well prepare for tissue, but nematodes float around, making difficult crash metal balls against the animal. I am talking about homogenization that can be used with tubes, and not driller systems. I have to isolate RNA from more than 300 samples and driller systems require to much time in our lab.
Has anyone experience with any of those machines (like Mixer Mills,FastPrep, or Bullet Bender) ?? Any advice or recommendation will be very welcome.
i don’t have any specific knowledge about any of the machines but the choice of worm disruption is to use glass beads (sand) because worms are small and slippery. do a google search (e.g.) for “elegans rna glass beads” to find articles that have the specifics on the material people used.
We use frozen worm pellets (dry ice). Then add 1ml of Trizon and homogenize using We use Wheaton overhead stirrer. You can get a lot of RNA. For example for L4 or adults it is easyly get 200 ug - 500ug for 1mL Trizon . I can send the protocol if you do not have one yet. email me kmassire@ucsd.edu
I know the Trizol method and we use to work with a similar machine that you, raymond, and of course works fine. I’m just trying to improve the method for us, in a way than we can process more samples at once. Apparently using the machines I mentioned, the system is not that efficient, even using glass beads like you recommended.
Probably it is a matter of vortex/shake time, rpm or liquid volume.
We’ve had success using the Trizol+glass bead method in a vortexer with a Turbomixer attachment (holds 12 microfuge tubes). Volumes are 0.1 ml frozen then thawed worm pellet, 0.3 ml Trizol, and 0.3 ml glass beads (Sigma, 425-600 microns) in 1.7 ml tube. Vortex 4 minutes at top speed in cold room. Add 0.7 ml Trizol, mix well by inversion, wait 5 minutes at room temp. Add 0.2 ml chloroform, mix 30 seconds by inversion, wait 3 minutes at room temp. Balance tubes and spin 15 minutes at 12,000 RCF. Transfer aqueous phase to new tube, add 0.5 ml isopropanol, wait 10 minutes at room temp, spin 10 minutes at 12,000 RCF. Aspirate supernatant, rinse pellet (may be glassy and hard to see) with 1.0 ml 75% ethanol, spin 5 minutes at 7,500 RCF. Aspirate all of supernatant (may require brief additional spin and aspiration) and resuspend in 0.1 ml DEPC-treated dH2O. Quantify [RNA] by spectrophotometer, and check quality by gel electrophoresis.
Good luck, and write back if you have additional questions or need more detail.