RNA extractions and bioanalyzer results II

I have done a couple trials of RNA extractions to figure out what is going wrong (see my last post on rna extractions).

All these were done using liver tissue which had been dissected and snap frozen on dry ice:

I extracted the first two and the results were not great, so I talked with J~ and realized some mistakes (too much tissue, no Beta mercaptoethanol, undergrads using my pipettes...). Then I did the next three rows to correct for those possible errors. I also changed how I'm washing the mortars and pestels: use bleach, then rinse with molecular grade water, use RNase-away and rinse with molecular grade water, then cover with tin foil and dry at 95c, and cool before use.

I won't post the entire bioanalyzer results, but here is a summary of the results:
I also did an experiment where S~ and I each extracted RNA side-by-side to make sure that I wasn't missing a step or doing anything else weird. S~ didn't notice anything, but the results (both hers and mine) were well below 8.

As everything up to this point was only mildly successful if that (none above RIN=8), I decided to do a comparison between completely fresh tissue and that same tissue that had been frozen. I sacrificed an animal and divided the liver into two parts - on went straight on dry ice and the other went directly to the extraction. The one on ice remained there until I finished the first extraction ~1.5hours.
I also realized that I have a second protocol that doesn't require the use of columns at all (it just uses the RNAsolv reagent), so I decided to run these next to each other. I followed the same protocol as above in regard to fresh and frozen tissue. The RNAsolv method ended up with 4 tubes per treatment. I only ran 2 of each on the bioanalyzer.

Results:



The two RNAsolv with fresh tissue didn't work at all and the other two were quite poor. I won't be using that protocol again. As for the Fresh vs Frozen tissue with the kit, it looks like frozen is actually better, though the difference is small. At least I have gotten two 8's though, that's looking up.

Not really sure what to do next. Committee members have said to just keep going until it's right, regardless of how long it takes. But I don't have anything to change, and furthermore I don't feel like I've done anything different even between the earlier 6.6 and this recent 8. The lab tech has said that she didn't realize that she was doing anything different when she suddenly started getting high RINs though so maybe there is still hope for me.

In unrelated news, it turns out that placenta has one of the highest incidences of RNases (along with pancreas). That doesn't explain poor quality liver RNA of course, but is something for me to keep in mind when extracting my placentas in the future...