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Old 06-08-2017, 05:51 AM   #1
SueFoltin
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Location: Ann Arbor

Join Date: Sep 2015
Posts: 11
Default RNA from FFPE vs quality for Illumina Seq

We have hundreds of RNA samples isolated from FFPE using the Qiagen AllPrep kit. I should point out that we have adapted the QIagen's protocol to add an additional RPE wash AS WELL AS an additional 80% Ethanol wash to remove as much salt as we can.

We are running into many problems in trying to get libraries generated using the TruSeq RNA Access kit from Illumina.

First problem: still have a lot of salt contamination.

Our 260:230 ratios range from 0.2 - 1.95. A majority of them correlate with concentration (the lower the concentration the lower the ratio) - but it's not 100% of the time.

Illumina is now recommending that our ratios be >2 and that we clean our samples.

I'm looking for recommendations for easy clean-up kits that won't further degrade our samples nor will I lose a significant amount of materil.


Second problem: DV200

Since our samples are coming from FFPE tissue, the process of tissue digestion and reverse-crosslinking leaves us with smaller than desired fragments.

Illumina is now recommending anything <30% not be processed - yet there is little we can do to improve this step.

I would appreciate any advise as I spend a lot of my time isolating RNA and DNA only then to not have it work with sequencing. It's very frustrating.

Thank you.
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