Has anyone used the DSN method (available on Illumina website - Duplex-specific thermostable nuclease)? I assume since it degrades highly-abundant transcripts, that it will prevent accurate quantitation, but I'm not sure to what extent. I have used ribominus in the past and it sucks (long protocol, not very efficient, low efficiency).
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
I don't have the data handy but according to Illumina it does not significant effect abundance of mRNA species. They have a plot comparing mRNA hits quantitated from a standard library prep (polyA selection) and a DSN library prep. They observed only a slight change for the most abundant transcripts.
The theory is that the conditions are tuned to hit the re-annealing curve before any mRNA derived cDNAs have had sufficient time to form duplexes.
Update:
Found what I was looking for. Slides 35-46 discuss the DSN normalization and slide #44 specifically addressed your question.
-
Originally posted by kmcarr View PostI don't have the data handy but according to Illumina it does not significant effect abundance of mRNA species. They have a plot comparing mRNA hits quantitated from a standard library prep (polyA selection) and a DSN library prep. They observed only a slight change for the most abundant transcripts.
The theory is that the conditions are tuned to hit the re-annealing curve before any mRNA derived cDNAs have had sufficient time to form duplexes.
Update:
Found what I was looking for. Slides 35-46 discuss the DSN normalization and slide #44 specifically addressed your question.
The following slide (#45) may show this, but does the "Total RNA" really include DSN? If so, why is the effect on the most abundant transcripts the opposite of what is on the previous slide?
As you said, the assumption is that only the most abundant transcripts are affected, which is not a problem anyway: who cares about housekeeping genes? I am still wondering if this would not affect a quartile-based normalization method for instance.
Comment
Latest Articles
Collapse
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...-
Channel: Articles
Yesterday, 07:01 AM -
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
51 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
45 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment