Hi Melanie,

Both the formalin and decalcifier you use can impact the RNA quality.

Maybe this article will help you:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440935/

Best,

Aga

Thanks Aga and Irfan! We are using EDTA for decalcification, but I was wondering if there is no protocol available without formalin fixation and without decalcification at all.

As much as I am aware, fresh frozen cryosections done without fixation and decalcification are doable, but usually require tungsten carbide knives and/or tape transfer system. Maybe check into those techniques?

Thank Irfan, but we do not aim to decalcify the bone completely, just to make it cutable.

Maybe these two publications are helpful for you:

Article Lack of Noggin Expression by Cancer Cells Is a Determinant o...

Article Biochemical Characterization of Major Bone-Matrix Proteins U...

Hi Melanie,

I think that it is possible to section mouse bone without decal, although we have never done it. You can search for plastic embedding and sectioning protocols, but they may also require a special blade for sectioning. To preserve RNA integrity, try 14% EDTA decalcification. You can do frozen sections afterwards, no need for paraffin embedding. Here's a link to a paper that looked at preserving RNA integrity...

Article Maintaining mRNA Integrity during Decalcification of Mineral...

Hope it helps:-) Let us know what works for you!

Hi all. I have had success using 10% EDTA in RNAlater for decalicification. I have presented a poster on this and did extensive research. It takes about 3 days (pH 5.2) but the RNAlater protects it for up to a week at RT. Keep it on a rocker. Your RNA will be much better this way, even if you formalin fix.

Ashley Fletcher Thanks a lot, I will try this.

Hi all. A good method for sectioning nonfixed and undecalcified bone is the Kawamoto Method.

Article Preparation of Thin Frozen Sections from Nonfixed and Undeca...