SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
will fragmentation buffer of mRNA-seq cut 200nt of my sample into small pieces? ziseputi Illumina/Solexa 1 09-02-2013 10:39 AM
Optimal fragmentation of bacterial mRNA Arvind Bhagwat Sample Prep / Library Generation 1 06-18-2012 08:33 AM
mRNA fragmentation kbushley Sample Prep / Library Generation 16 01-30-2012 06:29 AM
mRNA fragmentation for SPRIworks jowenMIT Illumina/Solexa 0 01-27-2011 01:21 PM
fragmentation buffer of mRNA-seq xile Illumina/Solexa 16 11-12-2010 08:12 AM

Reply
 
Thread Tools
Old 06-19-2014, 05:47 PM   #1
seqgirl123
Member
 
Location: U.S

Join Date: Oct 2008
Posts: 76
Default mRNA fragmentation RNA Seq

I am doing RNA-Seq, of mRNA by polyA bead selection. In the protocol I am using, it says to heat treat the mRNA for fragmentation. It does heat treatments at 94C at varying time lengths for varying size fragments. It then says to transfer the heated samples to a magnet so that the hot beads bind to the magnet. I wasn't watching the temperature drop back down on my thermocycler and by the time I did, the thermocycler was at 42C. I quickly transferred my samples to the magnet. But since the temperature dropped from 94C to 42C, will I lose yield of my mRNA? Or what kinds of problems will I run into? Is anyone familiar with different mRNA fragmentation treatments? Is 94C necessary?
seqgirl123 is offline   Reply With Quote
Old 06-23-2014, 05:17 AM   #2
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

The beads need to be magnetized while still hot so that the mRNA fragments containing poly(A) tails or internal poly(A) stretches will not have a chance to re-hybridize to the oligo d(T) beads. So in the situation you described, I think the biggest problem will be lower representation of these fragments in your final library. Re-heating the beads to 80 degrees and then placing them immediately on the magnet should allow recovery of these fragments without further fragmentation of the rest of the material.

I believe the temperature used for fragmentation depends on the divalent cation used in the fragmentation buffer- most library prep kits use magnesium (which is conveniently already present in RT buffer) so 94 degrees is the recommended temperature, whereas zinc-based fragmentation, such as that sold by Ambion, is performed at 70 degrees.
kerplunk412 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 12:37 AM.


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