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Old 12-12-2016, 11:54 AM   #1
kgoglin
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Default RNA QC: DNA contamination bad for rRNA depletion/mRNA capture

DNA contamination in an RNA sample can be detrimental to the rRNA depletion or mRNA capture assay prior to RNASeq library prep. We currently use the Bioanalyzer for RNA QC but it is not satisfactory for determining DNA contamination, unless it is obvious high molecular weight DNA. Sometimes an RNA with a good RIN score will still fail rRNA depletion/mRNA capture because there is DNA contamination that was not detected by the Bioanalzyer. Are there other types of RNA QC assays that could help us better determine whether or not we have DNA contamination in our RNA samples?
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Old 12-12-2016, 12:37 PM   #2
nucacidhunter
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I do not think that Bioanalyzer undetectable or even detectable DNA will cause depletion or mRNA capture faileur but it will affect depleted RNA-Seq data quality.

Quantification using Qubit RNA, ds and ssDNA reagents may hint presence of DNA contamination.
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Old 12-12-2016, 01:10 PM   #3
kgoglin
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Thanks for your response. We are not sure what the downstream sequencing would look like if we performed library prep on one of the DNA contaminated samples. Since the Bioanalzyer profile looked odd, we stopped there and DNAsed the Total RNA and repeated rRNA depletion. I posted below, a rRNA depleted sample from Total RNA before and after DNAse treatment. The profile after DNAse treatment is what we normally see as a "good" profile. The profile in which the Total RNA was not DNase treated, was odd.
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Old 12-12-2016, 03:15 PM   #4
nucacidhunter
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I do not know how rRNA depleted RNA profile should look like, but double depletion is expensive and not a good idea. The best practice is to do DNase treatment on total RNA extract before any downstream application. Genomic DNA contamination will be seen as high molecular fragments on RNA Chip. In your before treatment Bioanalyser trace if the peak at 100-150 bp are DNA they will be washed away during library prep and less likely will end up in RNA-Seq library. Generally DNA contamination will show up as higher alignment of reads to intergenic regions.
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