RNA degradation problem, only one band on the gel - (Jun/04/2012 )
Hi,
I am having a problem with the RNA integrity. I extracted RNA from e-coli using Phenol chloroform method, I managed to get good purity but the integrity does not look so good. I checked the integrity using non- denaturing formaldehyde gel. There was only One band at the bottom. I am not sure how to interpret it. Is it degraded? If it is, can you guys suggest anything to avoid it?
Does it have something to do with the number of cells for extraction?
Thanks in advance!
-Phaysalll-
That looks like degraded RNA fragments of ~100 nt in front of the bromophenol blue dye. For routine RNA integrity check, you don't need to pour and run the stinky formaldehyde gel. Regular native 0.8% agarose gel is good enough to see the 23S, 16S and 5S bands, if you add some 10-20 mM NaOAc in your loading dye (help holding up the RNA conformation for sharp bands) and run the BPB dye ~2 cm into the gel (banding becomes less sharp in long run).
When I purify RNA, I use clean tubes from an unopened bag (never autoclaved). Just make sure the bag says RNAse Free. Also I use filter tips. All the reagents are directly from bottles fro RNA purpose, making sure nobody introduce any spatule or any tip inside.
I use tissues and to homogenize the samples I treat the sonicator or the tearor before each sample with: water, EtOH 100%, water, NaOH 1M, 3Xwater and thiocyanate of guanidinum 4M. (make sure the water you use for the purpose is fresh from the miliQ system).This should prevent any contamination and external RNAse activity.
Good luck.
Jose Irimia on Wed Jun 6 00:24:05 2012 said:
I treat the sonicator or the tearor before each sample with: water, EtOH 100%, water, NaOH 1M, 3Xwater and thiocyanate of guanidinum 4M. (make sure the water you use for the purpose is fresh from the miliQ system).This should prevent any contamination and external RNAse activity.
Pun not intended, Jose, so many other people are doing exotic cleaning of their homogenizers for RNA extraction. Actually you don't need to worry RNase contamination in the homogenizer. Just image ... where would it have more RNases when you put the tissue in the homogenizer, the homogenizer or the tissue? lol. Just do normal wash, 10% Bleach rinse (to get rid of DNA/RNA cross-contamination), and then diH2O rinses, you are done. The real concern of RNase contamination is after the RNA is purified ... use clean tubes, clean tips, avoid touching the inside of the lid when opening the RNA tube, etc.