I extracted total RNA from animal tissue using the Qiagen RNeasy kit, however my RNA yield was extremely low and the 260/230 ratio was around 0.3. This is the protocol I followed:
- The animal cochleae were stabilised in RNA later.
- I crushed the cochleae in 600 ul of RLT buffer, vortexed it and centrifuged it at 10,000g for 10 minutes at 4 degree centigrade, and pulled the sup into a fresh tube. The cochlea is bony, this step has to be done so that we don't carry over the calcium and phosphates and clog the columns.
- After this I added equal amounts of 70% ice cold ethanol and then pipetted 700ul of this to the RNeasy column and followed the directions from the kit. I did perform the on column DNase digestion also.
However, our yields were extremely low, in the 20-30ng/ul range with 260/280 ratio's over 1.8, however the 260/230 ratio was around 0.3. I think that my RNA is still trapped in the columns and I need help extracting it.