I did RNA extraction using RNAzol (Sigma) on 1cm wound tissues but obtained this sticky opaque precipitate that I cannot dissolve in water. I have tried vortexing and incubating at 55-60 deg. I have performed the same method successfully on skin tissue.
During washing, it was quite hard to dislodge
These are the nanodrop readings for the soluble portion and a brief protocol.
A260/A280 : 1.42 - 1.53
A260/A230 : 0.07 - 1.43
1. Cut snap frozen wound tissue into small pieces on dry ice.
2. Homogenised in RNAzol (Sigma) using Qiagen Tissue Lyser II with a metal beat --> stored at -20 deg C.
3. Nuclease free water added to precipitate DNA and proteins --> centrifuge --> use supernatant
4. RNA precipitated using 100% isopropanol.
5. Washed 3x with 75% EtOH. The tube was spun down one last time and any excess EtOH was siphoned out. The tubes were then left to airdry for ~1-2mins.
After the apparent failure the 1st round, I took the remaining supernatant that I had from step 3, mixed it with equal volume 75% EtOH and purified it through a column. This gave better readings. However, the curve had peaks at 220nm and 270nm, most likely due to residual RNAzol.
A260/A280 : 1.37 - 1.74
A260/230 : 0.31 to 1.55
Now I have 3 sets of tubes
1. RNA from the 1st round
2. RNA from 2nd round
3. The "insoluble precipitates" sitting at -20 deg C.
I am hesitating to purify the RNA through a column as the yields are already very low. Hence would appreciate some insight.
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