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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:

  1. The animal cochleae were stabilised in RNA later.
  2. 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.
  3. 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.

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The ratio doesn't tell you the yield. The values of A260 correspond to the yield. The ratios just denote the purity. I am not aware of RLT buffer. It would be better for others too if you tell what that buffer is (is it similar to trizol ?). Just a suggestion: vortexing is generally not advised after cell lysis because it may shear the genomic DNA leading to some contamination. –  WYSIWYG Jun 12 '13 at 4:51
Why do you think it is still on the column, the problem could also be earlier in the process. Qiagen usually has a list of troubleshooting steps in their handbooks, I'd try to follow those. To elute from the colum I'd just try it again with water, and maybe incubate a bit longer. –  Mad Scientist Jun 12 '13 at 8:25
heated water also improves the elution –  WYSIWYG Jun 12 '13 at 13:43

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