SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Pippin Prep for size selection = win captainentropy Sample Prep / Library Generation 46 01-21-2016 11:00 AM
New, Completely Gel-Free, DNA Library Prep Protocol Bioo Scientific Vendor Forum 1 01-11-2012 08:49 AM
Pippin Prep for RNA? SageSigh Sample Prep / Library Generation 8 07-23-2011 08:41 AM
RNA prep for Multiplexed- Strand Specific RNA seq cmjluckey Sample Prep / Library Generation 0 02-21-2011 08:25 AM
Sample prep for RNA-seq from low abundance RNA chadn737 RNA Sequencing 7 06-15-2010 07:49 AM

Reply
 
Thread Tools
Old 07-14-2011, 02:38 PM   #1
shurjo
Senior Member
 
Location: Rockville, MD

Join Date: Jan 2009
Posts: 126
Default Pippin Prep vs. gel elution for RNA-Seq

I had a chance to do a side-by-side comparison of Pippin Prep (PP) and agarose gels (AG) for size selection during RNA-Seq library prep and would like to share these results with the community and get some feedback.

In the picture below, I show eight RNA-Seq libraries run on a BioAnalyzer HS DNA chip. The four sharp and high peaks are from PP while the four slightly lower and broader peaks are from AG. Equal aliquots of four adapter-ligated cDNAs were used for both platforms (so it's really 4 samples, then two techniques on each sample). Noticeably, the GS libraries have a lot more non-target material both above and below the peak, although all eight have a "bump" close to the upper size marker at 10380bp. A question: which of these would make the better candidate for submission to the sequencing center? I am leaning towards the PP as there is lesser background. Any advice from folks who are routinely using Pippin Prep for RNA-Seq?

Thanks,

Shurjo
Attached Files
File Type: pdf pippin_vs_gel.pdf (799.0 KB, 209 views)
shurjo is offline   Reply With Quote
Old 06-27-2013, 01:01 AM   #2
mbk0asis
Member
 
Location: Korea

Join Date: Dec 2011
Posts: 41
Default No way to remove adapter dimers?

The quote below is an old post from here in Seqanswers.
This guy compared two library size selection methods, agarose gel elution and pippin prep with his samples.
I like the sharp and narrow results of pippin prep, but adapter dimers still appeared at ~120bp. I thought most dimers would disappear when used pippin prep.
Has anyone tried pippin prep? Would somebody tell me how good it is?
And I'm planing to size select out 200~500bp. Would a broad range elution affect performance of the machine?

Thank you!!



Quote:
Originally Posted by shurjo View Post
I had a chance to do a side-by-side comparison of Pippin Prep (PP) and agarose gels (AG) for size selection during RNA-Seq library prep and would like to share these results with the community and get some feedback.

In the picture below, I show eight RNA-Seq libraries run on a BioAnalyzer HS DNA chip. The four sharp and high peaks are from PP while the four slightly lower and broader peaks are from AG. Equal aliquots of four adapter-ligated cDNAs were used for both platforms (so it's really 4 samples, then two techniques on each sample). Noticeably, the GS libraries have a lot more non-target material both above and below the peak, although all eight have a "bump" close to the upper size marker at 10380bp. A question: which of these would make the better candidate for submission to the sequencing center? I am leaning towards the PP as there is lesser background. Any advice from folks who are routinely using Pippin Prep for RNA-Seq?

Thanks,

Shurjo
mbk0asis is offline   Reply With Quote
Old 07-04-2013, 04:30 PM   #3
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 422
Default

The Pippen prep works just fine and reliable in our experience, but so can the gel extraction protocols. I would not think that the GE results from the previous post are representative. GE results can look a lot better - one might dissolve the gel pieces better at RT than at 50 degrees as most GE kit manufacturers recommend. Adapter dimers can also be removed with Ampure or equivalent beads. I don't see any signs of adapter dimer problems in the PDF above however.
If selecting a wide size range there can be potentially be problems with the collection well overflowing with the PP - I have not tried it, though - perhaps better use a low % agarose cassette then and ask their tech support?


Quote:
Originally Posted by mbk0asis View Post
The quote below is an old post from here in Seqanswers.
This guy compared two library size selection methods, agarose gel elution and pippin prep with his samples.
I like the sharp and narrow results of pippin prep, but adapter dimers still appeared at ~120bp. I thought most dimers would disappear when used pippin prep.
Has anyone tried pippin prep? Would somebody tell me how good it is?
And I'm planing to size select out 200~500bp. Would a broad range elution affect performance of the machine?

Thank you!!

Last edited by luc; 07-04-2013 at 04:37 PM.
luc 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 07:13 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO