4 replies to this topic

[jmerkin]

I am looking to do RT-PCR from animal tissue. I would like to grind it in with a mortar and pestle in liquid nitrogen first to break it up. I am concerned about contamination if I work with multiple samples. I can use porcelain mortars, but they are porous and I am concerned about RNA getting up in there. I can use glass mortars, but I am concerned about them shattering when I put the liquid nitrogen in.

Does anybody have any experience or thoughts on this?

[phage434]

A bead beater with disposable tubes and beads might be a better choice of technique.

[ivanbio]

I've used both successfully. In my experience the porcelain mortars work great for larger pieces of tissue (if you do not have much do not use these since you will lose a significant amount once your tissue is turned into a powder). Warning: make sure not to add more liquid nitrogen once your tissue has been turned into a powder and is dry. This may cause the powder to "puff" and you will lose part of your sample. Also work fast: once the tissue is a powder transfer it to the recipient you will use for extraction. This powder, when dry, will hydrate fast and make it harder to remove from the mortar. As for cross-contamination, I usually use multiple mortars at a time and once all have been used I stop to clean them all up before going through a new round of crushing.

Alternatively I suggest using a glass homogenizer. There is no need to use liquid nitrogen and they do a great job of homogenizing your sample.

[Ytje]

jmerkin, on Jul 14 2009, 09:55 PM, said:

I am looking to do RT-PCR from animal tissue. I would like to grind it in with a mortar and pestle in liquid nitrogen first to break it up. I am concerned about contamination if I work with multiple samples. I can use porcelain mortars, but they are porous and I am concerned about RNA getting up in there. I can use glass mortars, but I am concerned about them shattering when I put the liquid nitrogen in.

Does anybody have any experience or thoughts on this?

I work with gut tissue. First I cut the tissue up in a sterile petri dish with a sterile scalpel knife while the tissue is emerged in the lysis buffer (contains RNase inhibitors). After that I grind it (still in the buffer) in a tissue grinder (Potter-Elvehjem) at high speed. To finish it off I pass the lysate 10 times through a 21G neadle.
Works perfectly!

Ytje

[jmerkin]

Thanks for the suggestions. I've gone the glass homogenizer route before, but I'm going to be dealing with quite a number of tissue samples, upwards of 80. The sheer (no homogenizer pun intended) number is what concerns me. I think I may go with chopping up and homogenizing.