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I need to isolate RNA from Drosophila head. I basically chop the head off and first homogenize it with a homogenizer (similar to this: http://www.omni-inc.com/omni-tissue-homogenizer-th-package-p-11.html) and further homogenize it with 22G needle in TRIzol reagent. I use TRIzol RNA isolation for larvae brain a lot and have no problem with the method itself. However, with adult I got really low RNA concentration which is weird.

I thought that maybe I have problem with homogenization itself, or I overlook something (such as extra centrifugation or different kind of homogenization etc). Does anybody have experience with it? Thanks.

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You mean you have not a lot of RNA compared to whole larvae? Don't forget that in larvae a lot of gene expression is going on compared to the brains of developed flies. And the heads are a lot smaller, too. What does low concentration in terms of numbers mean? –  Chris Mar 10 at 16:17
    
I isolate RNA from larvae brain so not whole larvae. For example, from approx. 150 larvae after DNase treatment, I got more or less 120 ug RNA. For the very first time I have tried with adult. I used 220 adult heads and after DNase treatment concentraion of RNA was 52 ug. For me this result indicates a problem. –  golgicik Mar 10 at 16:25
    
Oh, then I misunderstood that. How is the quality of the RNA? –  Chris Mar 10 at 16:28
    
I measured with nanodrop and these are the ratios for adult RNA: 260/280= 2.17 and 260/230: 2.21 For larvae I usually get also more less these ratios. –  golgicik Mar 10 at 16:30
    
That sounds not bad for me, at least you have no quality problem. Do the heads dissolve completely? –  Chris Mar 10 at 16:35

1 Answer 1

It doesn't seem like a homogenization problem. Nonetheless you can try this:

  • Suspend the heads in trizol
  • Homogenize by passing through 30/31G needles (insulin syringe) up to 10-15 times.
  • Heating a little might help but I think it is unnecessary for these tissues.

Insulin syringe is much better than the hand held homogenizer. Freeze-thaw 1-2 times if you want (helps in homogenization).

During extraction you can add 5M LiCl (100µl) + Ethanol (1ml) to 400µl aqueous phase (instead of isopropanol), keep at -20⁰C for 1-2h before centrifuging. Alternatively you can add a little RNAse free glycogen to Isopropanol during precipitation.

PS: the liquid Nitrogen trick that GriffinEvo mentioned is for quickly getting heads and avoiding the pain of dissecting each head.

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