2 replies to this topic

[Liinda]

I need some advice please! I am planning to do Real-Time PCR on a mouse hypothalamic nucleus. I am concerned about my RNA yield and whether it is necessary for me to homogenise the tissue and how to go about doing it (this is mainly due to my concerns about my RNA yield). This is what I was planning to do:

-  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
   mixture)

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.

[Trof]

IMHO you need to disrupt the tissue, homogenisation can by done in Trizol by pipetting.
It depends much on the tissue type, maybe the brain would homogenise by just pipetting or you would need to pestle it in the nitrogen (but about the plastic micropestles that fit into eppendorf tube.. I originally thought it would be better because I can add Trizol right into it and don't lose any tissue, but found out that frozen spleen, liver or thymus is too hard to disrupt this way, so I abandoned it completely and disrupt in classic ceramic mortar and then add Trizol in it to collect the powder (and "wash" the pestle too)). 1mm x 1mm is AFAIK not such small amout to make problems if you do it this way, you just have to take care the piece wouldn't jump out of the mortar.

But the brain may be more soft, I would try to punch some random pieces of brain of required size and add Trizol and vortex/pipet up and down if it's possible to make homogenous lysate quickly. If so, you don't need anything else.
Just a note.. brain is considered an adipose tissue and that have consequences for Trizol isolation. Something about it can be found in the manual or elsewhere on forum.

I tried sonicating my samples (but of course you have to defroze them in something and that's first problem, usually RNAlater can be used, but previously nitrogen-frozen tissues require special type of RNAlater) but it didn't worked too well on my 3mm x 3mm samples, it took like 10 minutes to prepare one bacause of foaming and I wasn't sure I'm not damaging it.

[Liinda]

Thanks for all your advice Trof! Much appreciated!

If I can get away with pipetting up and down I will try that and am going to do some preliminary tests on yield etc this week but thought it's a good idea to get some advice first as well to try a couple of methods.

The fat in the brain may be a problem but that is why I am going to use TRI reagent - I read that organic extraction helps to remove more of the fat but it may block columns, plus it is cheaper to use TRI than columns and I will have lots of samples!

Thanks again!

PS I like your signature!