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  • #16
    Originally posted by ag4lm4 View Post
    Thanks for the reply. I did not fragment depleted RNA before cDNA synthesis. Still wondering why could not anuthing on Agilent?
    My guess: 90% chance you lost all your mRNA during an ethanol precipitation. 20 ng/ul is near the lower end of detectable DNA on a nanodrop. So it could be noise, or just some non-DNA/RNA stuff that absorbs a little at 260 nm.

    Unless -- some people do a nanodrop on a sample and then dilute their sample to the working range of a high sensitivity chip. Same issue as always with UV spec -- lots of stuff absorbs at 260 nm. But you might have plenty of sample but the dilution prior to running on a high sensitivity chip dropped it below the ability of the chip to detect. If that is the case, try running a 1 ul undiluted on a high sensitivity chip.

    --
    Phillip

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    • #17
      I made some RNA-seq libraries from partially degraded RNA from formaldehyde fixed FACS sorted cells.

      I used Ribo-zero to deplete the RNA according to the protocol, except for the final precipitation step I used AMPure XP beads and eluted in the "Elute fragment prime" solution in the Illumina TruSeq RNA sample prep kit. I fragmented the RNA and continued with the TruSeq protocol.

      There is a strange band at about 290 bp in the bioanalyzer image. Look at the first 6 lanes of the attached Bioanalyzer image. The peak is more obvious in the electorpherogram but our arcane software doesn't let us export the electoropherograms. So just take my word that it looks more worrisome in the electorpherogram.

      Does anybody have an idea what this source of this band or if it is a problem?
      Attached Files
      --------------
      Ethan

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      • #18
        Did you happen to run a pico chip pre-fragmentation? I don't think ribo-zero removes 5S/5.8S rRNA. If that did not fragment for some reason...

        I don't know, I think you stumped me on this one.

        --
        Phillip

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        • #19
          Unfortunately we do not have the RNA pico Bioanalyzer ChIPs. You think even if the RNA didn't fragment, the random priming would still give a broad range of fragments.
          --------------
          Ethan

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          • #20
            Yeah.
            I got nothing.
            Cut that band out, clone it and Sanger sequence it!

            Or, you could just do a gel cut on the pool you intend to send for sequence to get rid of that peak...


            Wait, look at this post. Sample 8 shows a phenomenon similar to yours.
            --
            Phillip
            Last edited by pmiguel; 05-03-2012, 03:13 AM.

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            • #21
              Originally posted by pmiguel View Post
              Did you happen to run a pico chip pre-fragmentation? I don't think ribo-zero removes 5S/5.8S rRNA. If that did not fragment for some reason...

              I don't know, I think you stumped me on this one.

              --
              Phillip
              We typically see >99.9% removal of 5.8S and >97.8% of 5S rRNAs in partially degraded RNA samples. Depending on the RIN of the original sample, I'd be concerned about over-fragmentation during library prep, but that's still unlikely to cause the peak at 290 bp in the library.

              Were the samples treated with DNase I after RNA purification?
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              • #22
                Interesting comments about rRNA removal with ethanol precipitation. Any experience with removing rRNA while trying to recover only small RNA ?

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                • #23
                  Originally posted by RNAseqer View Post
                  Interesting comments about rRNA removal with ethanol precipitation. Any experience with removing rRNA while trying to recover only small RNA ?
                  Eh? This makes it sound like the ethanol precipitation is removing the rRNA. The only ethanol precipitations discussed here were for sample concentration after binding of rRNA to ribozero oligos/magnetic beads.

                  --
                  Phillip

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