Usually I homogenize the bone fragments and proceed with conventional RNA isolation using TRI. But I find the purity of the RNA to be low. Which is the ideal method for storing bone samples and isolating RNA from the same? How good is the snap freezing of bones in liquid nitrogen?
See the attachment. may be this protocol use full to you.
See the attachment. may be this protocol use full to you.
You can also have a look at the web site Genomics Facility of Chicago University, there are severel different protocols proposed, best regards
stefania
http://fgf.uchicago.edu/protocols.php
We typically freeze whole bones (stripped of soft tissue) on dry ice or in liquid nitrogen. I then cryopulverize in a pestle and mortar on dry ice, in liquid nitrogen, before using RNA STAT60 to purify RNA.
I find that the RNA purity and integrity is good as long as the bones are kept very cold during the pulverization steps.
I have also stored bones at -80 ºC after freezing on dry ice and find that the RNA quality is still ok even after storing for several weeks.
Please let me know if you have any other questions, good luck with your work!
Hello
have tried many methods for this on various bone samples.
Collect the bone and immediately snap freeze it in liquidnitrogen. Store at -70 or 80 C until ready for use. Place bone in an RNase-free (soak with NaOH or SDS or autoclave or use RNAse-way) mortar and pestle and pour liqiud nitrogne in . Grind the bone into a powderwhile immersed in liquid nitrogen, keep topping up theliquid nitrogen. This keeps the bone frozen (preserving RNA)and makes it easier to grind when frozen as it shatters . Once powdered or at least in find pieces transfer the frozen fragments into a tube containing Trizol or GT extraction buffer and use a polytron homogeniser to further powder bone. It is important to completelypulverize the bone so as to break up all cells. Sometimes mouse bone is soft enough to skip the mortar and pestle step and go straight to homogenising it.It pays to cut up the bone into small bits with a scapelif you don't crush it in a mortar, otherwise the polytron can not churn up the large fragments. If you use Trizolmake sure you modify the protocol for firbous tissue as detailed by the manufacturer's recommendations. Yeild should be about 2000ug/ml from 200mg bone.Liz Smith
Good Luck.
Well I'm a bit biased because I work for the homogenizer manufacturer, but here is a link to a non-affiliated user of the Bullet Blender who gets great RNA yield and quality with their method.
Eric
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282642/
If we try all these methods (mentioned above) after removing bone marrow, these methods hardly work and do not show consistent results (RNA degradation after running on denaturing gel). Therefore, the RNA possibly we are getting is from bone marrow rather than bone itself. If any one has a good protocol where they are flushing out bone marrow still getting good RNA after confirming on denaturing gel, Please share. Thanks.
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