The DNase treatment I am using (Ambion, Turbo DNase-free) is completely degrading my RNA samples: I was testing the triplicate samples, where half of them were with the DNase treatment - and the other half without. The samples without had a RIN 7.0 - 8.9, while the samples with were so degraded that RIN could not be detected - and was clearly smeared when analyzing on Agilent Tapestation 2200. I want to use the samples for qPCR - but how do I remove gDNA without destroying the RNA?
I want to use the samples for qPCR - but how do I remove gDNA without destroying the RNA with the DNase treatment?
Any suggestions and experiences are very welcome!
I would suggest to compile some data that shows the RNA degradation and send it to the company. Their product is not behaving as it is supposed to and they should at least send you some new lot of DNase. You may also want to add EDTA to your samples to prevent RNA hydrolysis and make sure to leave the inactivation reagent undisturbed - I found accidentally carrying some over interferes with RNA quality. Moreover, during RNA extraction do NOT disturb the interphase, leave more of the aqueous layer behind to get better quality RNA.
This is an unusual result with Turbo. Spinning the Turbo Inactivation Reagent out of the DNAse treatment reactions at the end can be done for 3 minutes (instead of 1.5 minutes) at 10000 x g - did you perform that spin and remove the top ~72% of the reaction to collect your DNase-treated RNA? I have used Turbo for years now with no sign of RNA degradation. I have the feeling that you carried Inactivation Reagent over into your subsequent RNA analyses? A typical Turbo DNase treatment in my case is as follows (in 200 uL thin-walled reaction tubes):
Mix:
5 uL 10X reaction buffer
35 uL RNA sample
10 uL Turbo DNase (2 U/uL)
Heat for 30 minutes at 37C,
Add 5 uL Inactivation reagent,
Agitate/vortex for 2 minutes every 10-15 seconds,
Spin @ 10000 x g for 3 minutes
Remove the top 40 uL of the reaction (this is your DNase-treated RNA).
Discard the remaining bottom 15 uL (including the Inactivation reagent).
Has always worked.
I have had a similar experience when using the Qiagen DNase kit. I have noticed that I detect RNA up to 6 cycles later by RT-qPCR than DNA+RNA without DNase treatment. This suggests that DNase also destroys up to 98% of RNA (as my PCR targets a highly expressed gene, only a small fraction of all templates for qPCR would be DNA). One solution might be not to do RNase treatment, but run a qPCR as well as an RT-qPCR and to calculate transcript numbers as the difference between the two.
If this problem persists, you can try to design primers that span a genomic intron, e.g., span an mRNA exon:exon junction. This would eliminate the need for DNAse treatment as long as pseudogenes are not an issue for any of your qPCR targets...
Thank you for the good answers!
I have other co-workers that experienced the same degradation of their RNA samples by DNase treatment (different kits and different companies) - but did it anyway and detected the same later cycles as Christian described.
I think designing primers for introns is the best way to get around the degradation versus qPCR.
There have indeed been reports of DNAse reagents being contaminated with residual RNAses (left over from extracting the DNase enzyme from the source material) - and that could be a real and present danger with some DNase products... some DNAses (I believe either Qiagen's DNase or an Invitrogen DNAse) actually warn in their product literature that too long of a DNAse treatment of RNA with their product may result in loss/degradation of RNA.
Curious though ... I have checked my TRIzol-isolated total RNA (from lamb lung tissue) on a Bioanalyzer 2100 after Turbo DNase treatment and have gotten RIN values of 8.0 and above consistently. Perhaps Turbo is not consistent from batch to batch? Yikes.
The problem may be that your RNA is contaminated with RNAse and becomes activated under the conditions used for the DNAse treatment. I found this to be true for samples extracted from whole tissue (liver, others). Dnase treatment of these samples resulted in RNA degradation, but Dnase treatment of RNA samples from cell line culture were free of RNAse and did not show any RNA degradation after DNAse treatment under the same conditions. The problem was solved by adding RNAsin to the DNAse reaction for the liver RNA samples. RNAsin RNAse inhibitor is available from Promega and probably other suppliers and used according to the supplier's instructions.
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