I am isolating RNA of Agrobacterium and when I performed it for the first time it showed no degradation and now it is showing degradation. What can be the possible cause? I used Trizol method which we usually follow for RNA isolation.
@Zhi-Teng Is there any special method to isolate RNA from Agrobacterium. I do not want to go for kits. Is there any other protocol designed specially for Agrobacterium?
Which condition of the storage ypou use?
Is better than after made the RNA extraction, you convert it directly in cDNA. The cDNA permit you to conserve better and for more time your RNA samples.
No the degration occurs while extraction I think. When I isolate it I usually aliquot some of it to load on a gel and the rest I store it at -80degree cel. immediately. I take care of the temperature, cleanliness, fans are off, lab door closed and any possible contamination is taken care off by me. Can you please suggest anything which might be the likely cause?
I agrre with Isa.Try to clean up everything you use with DNAse and RNAse and use a new reagents. Clean with more attention the pipette, the support and the laminar and put all the staff inside the laminar with UV all night. If you go out from the laminar with the glove during the extraction, before to proceed wash the glove with ETOH and wait 5 sec to dry.
I agree with keeping environment clean while working on extraction. Also, I don't know how do you get samples of your bacteria and how fast it "dies" under exposure to air. The best way to prevent degradation of RNA during storage is freezing samples and kept them frozen until you add Trizol
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