SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
small RNA library between 50 to 250 nt foxytomato RNA Sequencing 0 10-24-2013 02:34 PM

Reply
 
Thread Tools
Old 12-06-2017, 10:42 AM   #1
fbarreto
Member
 
Location: San Diego

Join Date: Jan 2010
Posts: 15
Default crustacean small RNA library QC

Hi, everyone,

We're trying for the first time to prepare small RNA libraries for our crustacean model, and we're using the NEBNext small RNA kit. We just finished running the PCR products (before size selection) on BioAnalyzer. The trace looks very different than what I expected, but I only have the kit manual as reference. Mostly, I am confused by the very strong fragment at ~288 bp. The small RNAs are expected to be enriched at ~143-147, so my guess is that the peak at 150 is what I want.

Has anyone seen this? The concentration of the 150 peak is high enough for sequencing. So I am assuming I can just proceed with size-selection of that peak and be fine, unless someone has experienced this pattern and can comment whether my libraries failed and should not be sequenced.

Thanks for any feedback!
Attached Files
File Type: pdf small RNA library.pdf (291.5 KB, 18 views)
fbarreto is offline   Reply With Quote
Old 12-06-2017, 03:02 PM   #2
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,083
Default

150 bp is the target and it looks fine. The larger peaks depend on presence of other RNA species and would not be a concern. You might be able to correlate them with input RNA peaks if you have run a small RNA Chip.
nucacidhunter is offline   Reply With Quote
Old 12-07-2017, 09:09 AM   #3
fbarreto
Member
 
Location: San Diego

Join Date: Jan 2010
Posts: 15
Default

Thank you for your feedback! Looking at the TruSeq small RNA guide, it seems this is a common possibility, and may vary with organisms. I will proceed with size-selection.
fbarreto is offline   Reply With Quote
Old 12-13-2017, 10:21 PM   #4
sbarberan
Junior Member
 
Location: Santa Cruz, CA

Join Date: Feb 2017
Posts: 6
Default

How many cycles of PCR did you perform? We have seen a big peak ~280 when you over amplify libraries.

I think that these products at 280 could be 'bulge' products generated when you deplete primers, see (http://www.lsi.umich.edu/files/SmallRNACloning.pdf) and search for bulge.
sbarberan is offline   Reply With Quote
Old 12-14-2017, 05:22 PM   #5
fbarreto
Member
 
Location: San Diego

Join Date: Jan 2010
Posts: 15
Default

Hi, sbarberan,

We did 14 PCR cycles, with total starting RNA of ~700 ng. In consultation with our local sequencing core who does small RNA preps, they suggest these look fine, and we're proceeding with PAGE size-selection of the 140-160 bp bands. Fingers crossed.

I will update the thread when I get new BioA traces or sequences.

Thanks!
fbarreto is offline   Reply With Quote
Old 12-14-2017, 09:52 PM   #6
sbarberan
Junior Member
 
Location: Santa Cruz, CA

Join Date: Feb 2017
Posts: 6
Default

I'm sure if you PAGE size-select the libraries will be fine to sequence.

For next time I highly recommend that you use a gel-free library preparation kit. I work for Somagenics, and we just released our own small RNA library preparation kit that is gel-free and highly accurate (https://somagenics.com/realseq-ac). But there are other companies that also have gel-free library preparation kits for small RNAs.
sbarberan is offline   Reply With Quote
Reply

Tags
bioanalyzer, nebnext, small rna library prep

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 09:05 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO