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  • RNA Seq from FFPE samples

    Has anyone done any RNA Seq analysis from FFPE samples and if so can anyone offer any advice please on either the use of the mRNA-Seq Sample Preparation Kit or DSN normalization?

    Any advice would be greatly appreciated!

  • #2
    Originally posted by SallyH View Post
    Has anyone done any RNA Seq analysis from FFPE samples and if so can anyone offer any advice please on either the use of the mRNA-Seq Sample Preparation Kit or DSN normalization?

    Any advice would be greatly appreciated!
    We have not done any FFPE samples but I'll offer advice none the less. The standard mRNA-Seq library prep uses a polyA selection to remove rRNA; it assumes that your total RNA sample is very high quality (i.e. not degraded) so that you are pulling out full length mRNAs. FFPE samples are highly degraded so the standard mRNA library prep is not appropriate.

    When Illumina originally reported the DSN protocol they highlighted using it with FFPE samples since it works even degraded RNA. That is the way to go for your sample.

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    • #3
      I'm making some FFPE RNA-seq libraries right now.

      The RNA was purified with the Qiagen FFPE RNeasy minElute kit.

      For the libraries, I'm using the TruSeq RNA kit, except I've replaced the poly A selection with Ribo-Zero depletion. I did a test run of the protocol with one sample yesterday and it more or less looked ok on the Bioanalyzer. We'll see in some weeks what the sequence data looks like.
      --------------
      Ethan

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      • #4
        Originally posted by ETHANol View Post
        I'm making some FFPE RNA-seq libraries right now.

        The RNA was purified with the Qiagen FFPE RNeasy minElute kit.

        For the libraries, I'm using the TruSeq RNA kit, except I've replaced the poly A selection with Ribo-Zero depletion. I did a test run of the protocol with one sample yesterday and it more or less looked ok on the Bioanalyzer. We'll see in some weeks what the sequence data looks like.
        I don't do the wet lab stuff myself, meaning my knowledge here is theoretical so take it for what it's worth, but I question the effectiveness of Ribo-Zero on FFPE materials. Ribo-Zero, or similar rRNA depletion kits depend on targeted binding of specific oligos to pull down rRNA. If the rRNA is fragmented then only those fragments complementary to the oligos will be depleted, all of the other fragments will still be present in your library.

        This was one of the main advantages of the DSN method for rRNA depletion, it does not depend on any specific sequences of the RNA you are trying to deplete. It is solely based on concentration, and the relative concentrations of rRNA and mRNA should remain the same whether or not the sample is degraded.

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        • #5
          That is what I am most worried about, but this what epi-bio advertises:

          The Ribo-Zero™ rRNA Removal Kits (Human/Mouse/Rat) remove more ribosomal RNA (rRNA) from intact RNA, partially degraded RNA, and RNA isolated from formalin-fixed paraffin-embedded (FFPE) human, mouse, and rat RNA preparations than any other method. The Ribo-Zero kits set a new standard for rRNA removal for whole-transcriptome RNA-Seq.

          Benefits

          Removes >99.9% of 28S, 18S and 5.8S rRNA, and >95% of 5S rRNA from intact, and partially degraded, including FFPE-derived, human, mouse, and rat RNA preparations (Tables 1 and 2).
          Single-pass, 90- to 120-minute process yields RNA sufficiently depleted of rRNA for RNA-Seq (Table 2), random-primed cDNA synthesis, and other applications.
          Recovers mRNAs and ncRNAs for whole-transcriptome RNA-Seq.
          Preserves the transcript profile of the sample (Fig. 1 and 2).
          Recovers both the 5´- and 3´-end fragments of mRNAs and ncRNAs in a partially degraded RNA sample, including FFPE RNA.
          And experimentally backed hype:
          Blogger is a blog publishing tool from Google for easily sharing your thoughts with the world. Blogger makes it simple to post text, photos and video onto your personal or team blog.


          One thing is clear from what I have read is Ribominus form Invitrogen/Life Tech does not work as advertised. Ribo-Zero has gotten good reviews.
          --------------
          Ethan

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          • #6
            Thanks ETHANol, I happily stand corrected.

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            • #7
              I have done it both ways using Total RNA-seq bypassing the poly-A step with the TruSeq kits.

              In my hands the RiboZero works extremely well, but DSN can work just as well but if you are not experienced with it there is a higher chance of problems. We chose to use RiboZero for our FFPE studies due to its ease of use and reproducible reduction in rRNA.

              Also there is some debate whether to fragment or not. I chose to fragment to achieve uniform fragment size and we got very good 5'-3' coverage. We ended up doing 2x50bp and treated these as we would for degraded RNA samples for total RNA-seq.

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              • #8
                Also to echo ETHANol I have used both RiboMinus and RiboZero and there is not comparison. In fact RiboMinus should not even be an option they don't even touch the performance of the RiboZero beads for an rRNA reduction.

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                • #9
                  You'll get a lot of rRNA in your libraries using degraded FFPE RNA.

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                  • #10
                    Hi,
                    We have done RNA-Seq on RNA from FFPE using Ribo-Zero GOLD in combination with ScriptSeq (both from Epicentre).
                    On 240 months old FFPE samples we find 3-6 % of the reads mapping to rRNA - and much less (<1%) in younger FFPE samples.
                    Cheers,
                    Jakob

                    Comment


                    • #11
                      FFPE RNA using TRueseq RNA kit

                      Thanks for sharing your experience with FFPE RNA using TRueseq RNA kit.

                      For some specific reasons, we have to choose DSN-normalization method for my FFPE RNA library prepared with Trueseq RNA kit. Illumina told me that the and the DSN protocol won't work well with library prepared with Trueseq RNA kit.

                      Could you share more details, like input amount of adapter-ligated and enriched DNA, primers and condition used for final amplification?

                      Thanks in advance.

                      Comment


                      • #12
                        Re: Ribozero Gold and ScriptSeq on FFPE

                        Originally posted by JakobHedegaard View Post
                        Hi,
                        We have done RNA-Seq on RNA from FFPE using Ribo-Zero GOLD in combination with ScriptSeq (both from Epicentre).
                        On 240 months old FFPE samples we find 3-6 % of the reads mapping to rRNA - and much less (<1%) in younger FFPE samples.
                        Cheers,
                        Jakob
                        Jakob, I have been involved in a piece of work trying to do the same thing - but we just got massive adapter contamination in the final data. What size range was your FFPE RNA peak after RiboZero GOLD but before the ScriptSeq? Ours was between 25-500bp approximately, peak generally around 100.

                        Regards,
                        Hedley

                        Comment


                        • #13
                          FFPE samples with Trueseq RNA kit

                          I am working on the RANseq with FFPE RNA samples. I just noticed that the size of my dscDNA sample ranged from 100-200bp while after the end-repaire, A-tailing and enrichment, the size of my liberary ranged from 200-1000bp. Does anyone can tell me the reason and why is good for loading into the sequencer?

                          Thanks in advance!

                          Comment


                          • #14
                            Iahoman -- probably because your libraries are over amplified. There are a few threads on this topic here. Do a search. I do this to get the number of cycles right:


                            A completely different question...
                            What are people doing for fragmentation of RNA from FFPE samples? Do you decrease the incubation time at 94˚C? Do you fragment at all? Any thoughts on this.
                            --------------
                            Ethan

                            Comment


                            • #15
                              FFPE RNAseq

                              Thanks, Ethanol!

                              Comment

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