I am making RNA extractions with insect antennae and I would like to increase the yield of this process. For this, I use Trizol and I disrupt tissues manually with a pestle. Other disruption methods (beads or motorized pestle) gave lower yields.
Use liquid nitrogen to break your insect tissue easier. Add the trizol directly to the frozen samples. If you use bead beating on the powder with trizol you might get better yields. Crucial is not to let your sample to thaw without the trizol.
Use liquid nitrogen to break your insect tissue easier. Add the trizol directly to the frozen samples. If you use bead beating on the powder with trizol you might get better yields. Crucial is not to let your sample to thaw without the trizol.
Hi. For my part I used a polytron homogenizer to break insect antennae. Put your antennae in 450µl of Trizol then use the polytron at full speed during 30 seconde to break your tisues, and wash the polytron with the 650µl of remaining trizol directly in your sample) to collected back tissues that could have been bloked in the machine. Before the first use of the polytron and between each different tissues wash you polytron during 1 min at full speed in distilled watter then EtOH 70% then water again.
You might try using chitinase as long as you can stabilize the RNA at ambient temperatures.
I use the same proccess described by Erwan Poivet, but for between the uses, I wash it with NaOH 4M,distilled watter, EtOH 100% and then water again.
Froze your tissue (and metal beads already inside the tube) with liquid nitrogen, then use a MixerMill at full speed (30stokes/sec).
Liquid nitrogen is the solution. Freeze your samples and break it mechanicaly with a mixer mill if you can, or either with a mostar and pestle. The crucial point is to not let your sample thaw. The mostar have also to be frozen with liquid nitrogen.
Put immediatly your powder in a tube containing your Trizol reagent.
I use a kit to extract RNA from fly pupae. Whole pupae are homogenised in the lysis buffer with zirconia-silica beads in a bead-beater. The samples are then incubated with proteinase K for 10 minutes at 55oC with mercaptoethanol included. I use homogenisation under liquid nitrogen (as others have said) for DNA extraction from flight muscle or legs however this method resulted in degraded RNA in comparison to the bead-beater method described.
on the risk of blatant advertising, I suggest combining with an RNeasy kit. It could add greater stability and greater yield after liquid nitrogen. Use a filter or strainer to remove debris that would block the kit columns.
another risky Ad-post: Qiagen RNeasy kit already have the double column (the first one to remove debris and avoid column clumping)!
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