I'm using Ambion's TURBO rDNase and I think I'm seeing degradation of my RNA. Why is that?
Lane 1 = untreated RNA extract. 23s/16s intensity ratio = ~1.8
Lane 2 = ~0.5 ug of RNA, 37C for 30 min. 23s/16s intensity ratio = ~1.6
Lane 3 = ~0.5 ug of RNA, 37C for 30 min + TURBO + 1X Buffer. 23s/16s intensity ratio = ~1.0
This was a brand new TURBO kit.
And this was the second time I tried it. The first time was actually worse, but I forgot to record it. The 23s/16s ratio for the DNase treated RNA was like 0.6 and the bands were very faint.
I did not purify out/inactivate the DNase before running the gel, but I don't see how it could matter.
Hi Gen,
I have a few concerns with your question:
1) Since your lane1 sample is untreated (hence it is undiluted) and the sample in lane 3 is diluted (with 1X dilution buffer), it would definitely show the differences in the quantity (lane 2 , you have not mentioned about 1X buffer, so I suppose it is not diluted with the buffer, and the ratio is OK compared to 1.8 in lane 1) . Have you considered this dilution while loading gel ?
2) The samples are not purified and the buffer contains cations. So, in case your running buffer is heating up during electrophoresis, there is a chance of RNA degradation.
Personally I have never experienced RNA degradation during DNase treatment.
Best,
Akhil
I agree with Akhil Chameettachal . What you are seeing is a dilution of your sample and not RNA degradation due to DNase treatment. The approximate 23S/16S ratio is not necessary. One, it's an estimate at best. And two, degraded RNA will show up as a smearing of the bands.
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