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Old 11-26-2013, 12:48 PM   #4
thomasblomquist
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Location: Ohio

Join Date: Jul 2012
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Quote:
Originally Posted by KristenC View Post
Hi Tom,

I really enjoyed reading your paper! You touch on a couple of very important issues present in targeted RNA-sequencing.
I am currently working on a targeted RNA-sequencing assay, and am exploring possibilities for using reference genes to normalize the counts. Since your assay does not even need reference genes, I was wondering if you at some point looked into it and maybe dismissed it for certain reasons? Could you comment on why you moved away from possible adaptations of the more traditional RNA-seq approaches?

Kind regards,

Kristen
As to comment on why we moved away from traditional RNA-seq. Some preliminary data from colleagues using unsupervised biomarker identification in RNA seq libraries from different clinical samples was differentiating based on the DAY or the BATCH the RNA-seq library was prepped with!!! EEK!!!

This is not surprising as ligation efficiency is highly sequence dependent, and combined with fragmentation efficiency that can be highly dependent on protocols, we opted to develop a more focused and hopefully standardized approach that can answer focused hypotheses in our clinical samples. We work with small bronchial brushings with only 10-100 ng of RNA input, and we needed to be able to have reproducible and robust measurement on a couple hundred assay targets.

There are other reasons as well... But those were the big ones.

-Tom
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