RNA degradation after DNase1 treatment. - RNA degraded after DNase1 treatment. Any idea? (Feb/13/2008 )
Hi, everyone. Recently I am isolating RNA using Tri-reagent/Trizol. Some of the RNA showed DNA contamination, so I have run DNase1 treatment on these samples. I am using DNase1 from New England Biolabs. The protocol that I used is as follow:-
10ug of RNA
2.5ul of 10X DNase1 reaction buffer
2 unit of DNase1
DEPC-treated H2O to top up to 25ul reaction volume
The mixture is then incubated at 37C for 10minutes. Then 0.5ul of 25mM EDTA is added and heat inactivated at 70C for 10minutes.
The result that i get is weird, where the RNA degraded[18S and 28S bands not seen] while the untreated RNA remained intact.
I have try to lower the heat inactivation temperature from 70C to 65C, reduced the heat inactivation time to 5 minutes, and even adding in RNase inhibitor in the reaction, but yet the result is still the same.
Anyone of you have an idea on this?
Most RNA kits could not completely remove endogenous RNases and none, other than AquaRNA, irreversibly inactivate RNAses. Trace amount of the carryover RNases will degrade the purified RNA. That's why everyone says and knows that RNA is very unstable and very easy to be degraded.
A simple test to see if your purified RNA has RNase contamination is to add 1x DNaseI buffer (or any NEB buffer supplemented with 1mM CaCl2) to an aliquot of your RNA, incubate at 37*C for 10-15min, check on a gel along side the control RNA that is not incubated in the buffer. You could easily see the degradation of RNA if it is contaminated with RNases. We always do this to ensure RNA integrity before using them in any downstream analysis.
For RNA samples with minor RNase contamination and there is not tons of DNA, you could try RNase inhibitor and do the DNaseI digest in RT buffer, which does not have Ca+2 (Ca+2 can enhance RNase activity >1000 times), though the DNase activity is also reduced.