We need to get tissue free of RNA (and if possible DNA) of more than 500 pb long (for stupid but regulatory reasons). Tissues spend 2 weeks in PFA 4% also for regulatory reasons but it seems it doesn't break genetic materials. Do you have any cheap mean that will get rid of genetic materials (or at least highly fragment it) and remain these tissues suitable for IHC processing after that ?
I think you can try to use DNase or RNase on the slides before staining.
PFA treatment will not harm nucleic acids, it only crosslinks them with their surrounding proteins. I would also think about DNAse/RNAse treatment, but I have no idea how feasible this is. Are there any colleagues you can ask about this, which had to deal with this problem alreaedy? Or is this a new regulation so nobody has experience so far?
This is a new regulation and we have to find a way to apply it before paraffin embedding ... I already thought about RNAse and DNAse but I guess it would be very costly to apply it on every organs that we need to analyse and I am not sure of efficiency as the penetration of the enzyme may not be very good.
I also thougt about gamma irradiation ? Do you have experience of that ?
You have the problem of getting the enzymes to the DNA, which will be at least difficult to do. Thats why the treatment of the slides was mentioned, since the cells are cut open there. The problem with removing or breaking DNA/RNA without damaging anything else will be quite difficult.
Can I ask why this regulation was introduced?
So basically what your problem is: you have an antibody which stains RNA but you need to detect proteins (only)? Because defining the problem could help to find a solution.
As I understand Kevins problem he need to eliminate all DNA/RNA (or at least break down below 500bp) in his sections due to some stupid regulations.
We used sodium hydroxide (alkali) to destroy RNA & acid (0.2 M HCl) to get rid of DNA in gels, so i guess the same could be titrated for use in tissue samples
Thanks for all your answers. To answer Christian's question, I am working with Henipaviruses in a BSL-4 laboratory so we have to be sure that the virus is completely destroyed and that is why the organs spend two weeks in PFA. Since 1st of July 2012, a new french law was introduced to regulate genetic materials from dangerous viruses and consider that every fragment of more than 500 bp has to be declared and cannot be moved without special authorisations. It's completely stupid as it is not infectious at all and totally safe to handle but still ...
Apparently we found that Bouin liquid which is also a fixative destroys quite well genetic material (but it is also aggressive towards proteins...). We will try to let the organs one day in this fixative to see if it destroy genetic material.
Ok, if you deal with BSL-4 material, I understand why there has to be extra safety. I was only thinking about ordinary tissues. But you of course never know, what ideas regulating agencies have.
There are two variations of this solution, which might be interesting as well: Gendre Solution and Hollande Solution.
- Badges
- Science method