- use a mouse brain matrix to cut a 1mm thick brain slice containing the nucleus
- place slice on a slide and use a 1mm guage tissue punch to isolate the nucleus and place in a tube on dry ice
- keep tissue in -80oC freezer until all samples have been collected, then carry out extraction
- use TRI reagent (essentially the same as TRIzol - phenol and guanidine thiocyanate based organic extraction solvent
I have seen that people use glass homogenisers or plastic pestles to grind up tissue before or after adding TRI reagent, tubes with ceramic bead matrix, sonicators or pass the tissue in TRI reagent through a syringe with a small gauge needle attached. But as I am using such a small amount of tissue I am worried. I suppose though people get enough RNA even from laser microdissection so maybe I shouldn't worry but there are only a few months left on the grant!
So, really my questions are:
- if you have experience of doing something similar, do you think I'll get a decent yield of RNA?
- do I have to homogenise the tissue using some piece of equipment - if so what works well and is cost-effective? Can I just vortex the tissue in TRI reagent?
- does the equipment used to homogenise the tissue reduce the yield due to adherence to the equipment?
I would really appreciate some advice if you have experience in this!
Thanks a lot!
Edited by Liinda, 29 November 2011 - 03:13 AM.