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SueFoltin 06-08-2017 05:51 AM

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.

lexogen 06-08-2017 12:53 PM

3' mRNA-seq gene expression profiling by QuantSeq
if you think gene expression profiling by a 3' DGE kit that can target heavily degraded, polyadenylated RNAs is applicable for your project, then have a look at the QuantSeq kit from Lexogen.

Reproducible, high-quality gene expression values can be generated from degraded or FFPE-extracted RNA with low DV200 values, and there is even a corresponding award program that might be of interest to you. The kit price itself starts from USD 19.80 and includes read data analysis on the BlueBee platform.

Regarding the contaminations in your sample you might want to look into bead based purification protocols that can be applied in the 96 well format to your large number of samples. Alternatively, the QuantSeq kit accepts very low inputs, i.e. from 10 ng for degraded RNA, hence it might be possible to simply dilute your samples (and the contaminations) accordingly.

For further info please contact

jteeee2 06-08-2017 01:37 PM

For column cleanup of your already extracted RNA, I'd recommend the Zymo RNA Clean and Concentrator-5 workflow. Cheap, easy to use, and allows a smaller elution volume than compared to others (i.e. Qiagen).

As far as your DV200 issues, you are correct that there is not much you can do to fix that. We have found that it's important to do some upfront testing on the different kits out there to find one that produces the best quality/quantity of RNA. We have found the Qiagen systems to be the worst when it comes to extracting longer RNA molecules (DV200 values always lower when we use the Qiagen workflows as compared to other kits on the market). If you are open to trying new extraction techniques, I would point you to a new company that spun out of Stanford. They supposedly have a kit that is better suited for FFPE RNA extraction that what is currently on the market.

The RNA Access kit will generate libraries from material with a DV200 <30%. You may need to include additional PCR cycles in the initial library prep to generate enough material for the hyb/capture steps. However, these libraries will inevitably show lower complexity and a very high duplication rate...both very common with FFPE RNA Seq libraries.

SueFoltin 06-09-2017 05:33 AM

Lexogen and jteeee2:
Thank you so much. I really didn't expect anyone to respond as most don't work with FFPE. These are the only tissue source we tend to have issues with and having the collective share their experience certainly goes a long way to help.

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