Not an easy task, I must say...

First of all: how do you homogenize your samples? I would strongly recommend precutting the tissues and then using Dounce tissue grinder (all with some sort of RNA-protective solution, like for instance RNA Later).

Second: try an old and dusty but still a good method of phenol/chloroform extraction. TRIZOL is also based on this method but in Addition, it contains heavy salts.

Third - have a look at this patent description, perhaps this can help: http://www.google.com/patents/EP2625271A2?cl=en

Lastly, have a look at the references, such as http://www.plosone.org/article/fetchObject.action?uri=info:doi/10.1371/journal.pone.0038587&representation=PDF

Good luck!

Thank you very much Agnieszka for your response.

As I mentioned we use the TissueLyser with the stainless steel beads from QIAGEN at different speeds and times.. Larvae are extremely tiny... About 1mm.. So you can't grind/precut anything, you can barely see the larvae in the tube. 

Literature is helpful, but most of the time they mention "isolation was performed using manufacturer's suggestions" or so. Which is not that helpful, and most of the times that's what everybody is starting with. 

Thank you, also, for the links attached. The link to the patent looks very interesting, even though they are mostly using the invention for DNA extractions. We do use a similar lysis buffer recipe in the lab for DNA extractions. 

Thank you once again for the assistance !

Hi, 

I am isolating RNA, after adding glycogen and isopropanol, centrifuge for 10minutes 12000g, I can't see RNA pellet ? 

Note: I incubated RNA with isopropanol for 25minutes at room temperature.

any suggestion plz why i can't see RNA pellet ? and if i should continue my experiment or not ?

Thank you.

Dr. DG if you give some further info on your tissue/cells/experiment we might be able to assist you. 

I wouldn't leave the RNA at RT for too long; even though if your solution is RNase free it should be rather stable, but I wouldn't risk it.