SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
DSN normalization henry.wood Sample Prep / Library Generation 3 11-19-2014 12:36 AM
RiboMinus yields pmiguel Sample Prep / Library Generation 6 06-21-2012 08:04 AM
Illumina vs Evrogen DSN anar RNA Sequencing 1 08-17-2011 07:59 AM
DSN experts? whw Sample Prep / Library Generation 3 08-14-2011 05:29 PM
Bioanalyzer profile after ribominus treatment sanush RNA Sequencing 0 03-05-2010 10:25 AM

Reply
 
Thread Tools
Old 10-25-2010, 09:51 AM   #1
daughart
Junior Member
 
Location: Philadelphia PA

Join Date: Aug 2010
Posts: 5
Default ribominus vs DSN

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).
daughart is offline   Reply With Quote
Old 10-25-2010, 12:31 PM   #2
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,177
Default

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.
Attached Files
File Type: pdf 2010-06_sq_03_lakdawalla_transcriptome_sequencing.pdf (4.65 MB, 111 views)

Last edited by kmcarr; 10-25-2010 at 12:36 PM. Reason: Add file
kmcarr is offline   Reply With Quote
Old 10-25-2010, 07:36 PM   #3
ScottC
Senior Member
 
Location: Monash University, Melbourne, Australia.

Join Date: Jan 2008
Posts: 246
Default

The other main benefit being that you can analyse degraded RNA samples because you don't have to use a poly-A selection step (they discuss using RNA extracted from FFPE tissue).
ScottC is offline   Reply With Quote
Old 10-26-2010, 01:10 AM   #4
steven
Senior Member
 
Location: Southern France

Join Date: Aug 2009
Posts: 269
Default

Quote:
Originally Posted by kmcarr View Post
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.
Thanks kmcarr for providing this document. I had seen this slide too, but now i realize that on the y-axis there is still a polyA selection going on. I am curious about what would give polyA (-DSN) vs. total (+DSN).
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.
steven is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 12:44 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO