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Old 12-11-2017, 09:20 AM   #1
arctan
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Default genomic DNA contamination before RiboZero and ScriptSeq

This will be my first time making RNA-seq libraries. I have the RiboZero (for bacterial RNA) and ScriptSeq library prep kit.

I prepared bacterial total RNA and did on-column DNase treatment. NanoDrop 260:280 ratios are 2.0-2.2. The RiboZero protocol recommends quantifying RNA concentrations using Qubit, so I used the RNA Broad Range assay for that. For kicks, I also tried the dsDNA High Sensitivity assay on the same samples.

I am getting about 6-8% genomic DNA contamination compared to the concentration of RNA, but naively, I was expecting much less.

Has anyone done similar DNA vs RNA Qubit measurements for their samples for library prep, and what ranges do you get?

Also, how bad is my 6-8% contamination for RiboZero and subsequent library prep, since the genomic DNA will still be present after rRNA depletion?

Since a RNA purification step is required after RiboZero, perhaps I can proceed with RiboZero first, then do an additional DNase treatment immediately after, and then proceed with the RNA purification step? Or should I not worry about the 6-8% of DNA?

Any recommendations or advice on how to proceed would be much appreciated! I am completely new to this...
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Old 12-12-2017, 09:23 AM   #2
pmiguel
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Hi arctan,
You might want to take an aliquot of your sample, heat it to 70 oC for a few minutes to denature it's secondary structure, snap cool and then retest for the presence of genomic DNA.

Although the Qubit dsDNA High Sensitivity fluor detects primarily double stranded polynucleotides, I do not think it distinguishes between double-strand DNA and double-strand RNA. So any secondary structure in your RNA can read as DNA.

To the extent your RNA has long stretches of double stranded regions, the 70 oC treatment will not be sufficient to denature it.

Another alternative would be to take an aliquot of your RNA and treat it with a very effective RNAse, maybe if you can find a mixture that can degrade both ssRNA and dsRNA. Then check this aliquot for DNA.

--
Phillip
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Old 12-12-2017, 10:55 AM   #3
jteeee2
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That material will be fine for a total RNA Seq approach. I wouldn't get concerned unless that value is above 10%. Also agree with pmiguel that the Qubit value can be misleading.
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Old 12-13-2017, 10:58 AM   #4
arctan
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Thank you both for the helpful replies!
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