PCR Sample Prep - (Jul/14/2009 )

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?

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

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.

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

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.