RNA.seq FFPE

[Nichren]

Hi All,

I'm fairly new to RNA.seq and newer to RNA.seq of FFPE tissues. And thought I'd tap into the knowledge base here.

We have a number of RNA samples derived from FFPE tissue, they have DV200 scores above 50% and 1ug or so total yield. We are looking to get as much information from them as possible and I was thinking of using the Illumina RNA Exome kit for library prep or another capture method to look at isoform changes.

Or should I just be looking at ribo-depletion and sequencing deep?

[nucacidhunter]

You have enough quantity and relatively good quality RNA for a FFPE sample. Ribo-depletion will be good choice as it is open for transcriptome wide discovery and sequencing cost with NovaSeq is also relatively low.

[Nichren]

You have enough quantity and relatively good quality RNA for a FFPE sample. Ribo-depletion will be good choice as it is open for transcriptome wide discovery and sequencing cost with NovaSeq is also relatively low.

Thinking TruSeq Stranded Total RNA, to get as much information as possible, and yes they are surprisingly good samples.

Any experience with this library prep kit and FFPE RNA, nucacidhunter?

[nucacidhunter]

In theory TruSeq can be used but I have not seen any convincing evidence from Illumina using this kit for FFPE RNA-Seq. Instead, they have focused more on targeted approaches such as RNA exome (formerly RNA access). This paper also indicates poor performance:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170632

Other supplies such as NEB (NEBNext Ultra II Directional RNA Library Pre) or Clontech (SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian v2) have published tech notes using their kits and worth considering.

[Geneus]

Quick question for the OP...what method did you use for your extraction from FFPE and how many curls/slides did you use to obtain 1ug of RNA? If these were slides did you perform any macro-dissection? The devil's in the details.