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Old 06-08-2017, 01:37 PM   #3
Location: Illinois

Join Date: Oct 2014
Posts: 37

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.
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