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?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
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.Attached Files
Comment
-
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.
Comment
Latest Articles
Collapse
-
by seqadmin
Innovations in next-generation sequencing technologies and techniques are driving more precise and comprehensive exploration of complex biological systems. Current advancements include improved accessibility for long-read sequencing and significant progress in single-cell and 3D genomics. This article explores some of the most impactful developments in the field over the past year.
Long-Read Sequencing
Long-read sequencing has...-
Channel: Articles
12-02-2024, 01:49 PM -
-
by seqadmin
The field of immunogenetics explores how genetic variations influence immune responses and susceptibility to disease. In a recent SEQanswers webinar, Oscar Rodriguez, Ph.D., Postdoctoral Researcher at the University of Louisville, and Ruben MartÃnez Barricarte, Ph.D., Assistant Professor of Medicine at Vanderbilt University, shared recent advancements in immunogenetics. This article discusses their research on genetic variation in antibody loci, antibody production processes,...-
Channel: Articles
11-06-2024, 07:24 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 12-02-2024, 09:29 AM
|
0 responses
124 views
0 likes
|
Last Post
by seqadmin
12-02-2024, 09:29 AM
|
||
Started by seqadmin, 12-02-2024, 09:06 AM
|
0 responses
47 views
0 likes
|
Last Post
by seqadmin
12-02-2024, 09:06 AM
|
||
Started by seqadmin, 12-02-2024, 08:03 AM
|
0 responses
38 views
0 likes
|
Last Post
by seqadmin
12-02-2024, 08:03 AM
|
||
Started by seqadmin, 11-22-2024, 07:36 AM
|
0 responses
67 views
0 likes
|
Last Post
by seqadmin
11-22-2024, 07:36 AM
|
Comment