Hi,
I am currently dealing with extremely low amounts of environmental microbial RNA (typically
You can use thermofisher's DNA-free™ DNase Treatment & Removal Reagents
DNAse or heat deactivation most probably would not be the reason for RNA degradation. There must be something else. 65 degrees are nothing and generally used in many RNA preparation protocols.
It must be contaminated reagents or equipment which might have caused RNA degradation. Also, I wonder how you might be able to access/interpret integrity of RNA based in qubit?
Abhijeet Singh I just measured the amount of RNA before DNAse treatment and after. The numbers drop 80-90% in the few samples I used, so that's why I was suspecting the thermal treatment. But that's a nice point, maybe pipets/air/tips are contaminated with RNAses and I didn't clean thoroughly enough?
I guess I should try the same procedure without thermal treatment and then - if that doesn't change anything - do a very thorough cleanup of the pipets and play around in the "clean" room with fresh tips. Thanks for the food for thought!
Bashiru Garba Thanks, just ordered that kit a few days ago, but I read that some RNA loss happens with this kit too and I have very low amounts of input material, so I am preparing for the kit's potential failure ahead ^^'
Artur Zaduryan
1. measured the amount of RNA before DNAse treatment and after
>> I imagine between measurements, there are cleaning steps involved. Each additional wash would cause some loss, doesn't matter what type of columns or kits are used. If you did not clean DNAse and buffer from the RNA samples, they are probably should not be used for RNA measurements.
>> about 90% times, RNA problem occurs due to user/handling related problem. If you are not working with RNA sample frequently, frequent change of gloves and RNAse cleaner solutions must be used. Cleaning of all surfaces, pipettes, centrifuge etc are mandatory and prerequisite for RNA work. Forget your phone if you are using it meanwhile. Avoid touching skin surfaces with gloves while working.
3. I guess I should try the same procedure without thermal treatment and then - if that doesn't change anything - do a very thorough cleanup of the pipets and play around in the "clean" room with fresh tips.
>> You may do as you prefer, but cleaning should never be a trouble shooting step. It is a prerequisite.
Alright, great. You can also make efforts to concentrate the RNA by targeting rich environmental samples. Best of luck
your measurement of RNA sample before and after DNase treatment is not accurate, I won't use it as evidence that RNA's partial digestion.
Artur Zaduryan I had similar issues, but my samples had more RNA concentration that yours. My RNA suffers degradation at the DNAse heat inactivation step even adding EDTA. Probably RNases from the sample acting at this step were the cause.
The Solution was to do a phenol:chloroform DNase inactivation after de DNase treatment, but the loss of material is apox 50%
André Lasalle Thanks for your input! On a slightly different note, what gel/stain are you using for your RNA gel?
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