RNA extraction from whole tissue

[moerea]

Hi,

I'm in need of doing some RNA extraction from tissue from adult zebrafish and I was wondering how many fish people usually pool in order to get a decent amount of RNA and good RNA quality. People in the lab have been pooling 10 tissues (10 x heart, 10 x spleen and so on) for extractions and I don't know if that's a fair amount or if I should go higher/lower.

Also are there any good advise in getting good quality gill RNA (how many should be pooled, etc)? We've always been having issues with that in the lab.

Thanks in advance.

[blanco]

Hi, I am looking for the same info as the original poster. Any experienced RNA extracter out there?

thanx
blanco

[danfive]

Hi, I use to work at a veterinary diagnostic lab (Aquatics), and used the following protocol to get the best rna possible --for diagnostic screening purposes.

[Only registered users see links. ]

Note the recommendation of 350mg to 1 mL homogenization buffer (PBS). There really is no lower limit, properly done the trizol extraction will get any rna introduced.

[danfive]

Regarding the gills, here's the trick again from the diagnostic lab protocols,

Originally Posted by danfive

I worked with PCR from gills, as well. They are not very difficult but you should consider that the gills may contain a lot of blood and the iron in the hemolymph/globin is a PCR inhibitor---solution to problem: cut small sections of gill (i.e. 0.1g) and soak in pure water for 20 minutes. Take your tissue and just about any DNA extraction procedure will work and give you good quality DNA from there. To be extra secure you can use a Clontech Chromaspin Column-100 to purify your DNA a step further.

Same trick for RNA, cut into small fragments and soak 20 min, then proceed with homogenization and Trizol extraction. Won't need the clontech column for additional clean-up.