Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq sample prep from blood epibio Vendor Forum 0 03-20-2013 10:16 AM
Small RNA sample prep.... Sinha_Ashis Sample Prep / Library Generation 2 05-01-2012 12:49 PM
Small RNA Sample Prep v1.5.0 jujuhi Illumina/Solexa 19 11-21-2011 10:10 PM
RNA-Seq sample prep... SWP Sample Prep / Library Generation 2 07-08-2011 03:01 PM
Sample/library prep of DNA and RNA in a metagenomic sample chrisaw01 Metagenomics 1 05-05-2011 01:59 PM

Thread Tools
Old 08-03-2016, 01:07 PM   #1
Junior Member
Location: Rhode Island

Join Date: Feb 2014
Posts: 9
Question Sample prep from different cells when amt of RNA is vastly different...?

I'm doing an RNA-seq experiment comparing transcript expression across different stages of development.

My first stage contains about 60-fold less total RNA than the final stage. Most of the library prep protocols suggest starting with a certain concentration of RNA. If starting with 1 ug of total RNA input, the late stage sample would require only 1 ul of RNA, but the first stage would require 60 ul. I could dilute the highly concentrated RNA, or make the less abundant RNA more concentrated by using an increased sample size, but I'm worried about any bias this might introduce.

When comparing samples with vastly different amounts of RNA, what's the correct approach? Should all RNA just be diluted to a certain concentration? Or, should the starting RNA be based on volume (ie: 1ul of sample A, 1ul of sample B, even though total RNA concentrations would be very different).

Does a volume based approach more accurately capture the true picture? Does diluting to a specific total RNA concentration perhaps obscure less represented RNAs?
sjeschonek is offline   Reply With Quote
Old 08-05-2016, 09:22 AM   #2
Senior Member
Location: Bay Area

Join Date: Jun 2012
Posts: 109

Most input related bias (Every library prep method is biased in one way or other, but usually consistently biased as long as you stay with the same protocol) in RNA-Seq isn't so much bias but noise. As you have fewer and fewer molecules going into the prep, the Poisson noise from the presence or absence of transcripts and library prep inefficiencies start to dominate the lower expressing genes and destroy your ability to get statistically relevant DE.
You'll be best off sticking with the same kit/protocol for all samples and trying to get as much RNA, up to the recommended amount, for each sample into the library prep. If you can't concentrate your early samples enough for this, it might be advisable to choose a protocol specifically intended for lower input. They generally have higher efficiency molecular biology that reduces the aforementioned noise issue.
cmbetts is offline   Reply With Quote

library bias, rna-seq advice

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 01:59 PM.

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