RNA isolation from bone - (Feb/21/2001 )
Can anyone help me with setting up a protocol for extracting RNA from mouse bone tissue?So far I've found a lot of protocols for isolating RNA from soft tissues which get frozen and ground in a mortar.My estimation is that bone is a bit harder to pulverise. Therefore I was hoping for a recipe that makes the bone softer without damaging the RNA.
Thanks in advance,
I have tried many methods for this on various bone samples.
Collect the bone and immediately snap freeze it in liquidnitrogen. Store at -70oC 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.Goodluck. Yeild should be about 2000ug/ml from 200mg bone.Liz Smith