I am investigating the transcription levels of some genes in the brain compared to the body of zebrafish. How can I extract RNA from the brain (or the head) and the rest of the fish separately? Usually, the dissection of organs takes time which is not good for the isolated RNA quality. How can I overcome this problem?
Thanks a lot for your help
Hi Remon,
For having done mouse testis RNA extraction - following dissection from adult or FACS sorted germ cells - I can tell you that for most applications (both long and small RNA profiling) as long as you keep your dissected sample on ice or immerse them in Trizol right away you should be fine.
Alternatively you could try to collect brain and control tissue for each animal in epi tubes and snap freeze them one by one in liquid N2. Once you're done, you can pool them and process them all at once - like douncing them in trizol.
Hope it helps.
Cheers
Antoine.
Commonly RNA isolation kits are available from Qiagen or invitrogen.It is as a common procedure which you can isolate RNA from other tissues.
Hi Remon,
Like Antoine Molaro suggested at the time of dissection you can cut zebrafish head with a bisturi, put it in an eppendorf tube and freeze it in a container with liquid nitrogen . It is a rapid process. Then you can extract body organs and preserved them on RNAlater. For RNA extraction common RNA isolation kits are available. For example i use “illustra RNAspin Mini Kit” (GE Healthcare) to extract RNA from zebrafish gonads.
Joana
Many thanks for all of you. But my problem is not the RNA extraction itself but how to prepare the biological material without affecting the RNA quality. To separate the brain or the whole head of the embryos it takes time which might lead to RNA degradation.
So I was wandering if there is a protocol to isolate the brain or the head rapidly and under conditions that keep the RNA intact
RNA quality shouldn't be a major concern. Anaesthetisation may take 1-2 mins followed by dissection of the brain which takes 2-4 mins depending on skill. This is a reasonable amount of time. If you have access to a cooling block, perform dissection on them. Or else, try dissection on a pre-cooled wax block. If you are still paranoid, you can even try to perform the dissection in RNAlater rather than PBS. Then check the quantity and quality of your RNA on a gel/Bioanalyzer/Tapestation etc. If the ribosomal bands are clear and has a 2:1 ratio, you should be fine. As a final note, even if there is some minor degradation, if all your samples are treated the same way, they should be comparable.
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