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  • Help with determining RNA quality for RNA SEQ

    Hi All,

    I'm new to RNA-Seq, so I'm not that familier with RNA quality checks for RNA seq. I have 6 total bacterial RNA samples, that I want to carry for rRNA depletion using the RiboZero kit. Before doing that I ran the samples on agarose gel and got a strange result (please see attached). It seems that all the samples have a huge band around 100bp.. what this means? is that degraded RNA? or maybe DNaseI degraded DNA leftovers?
    Any help interpreting this gel will be highly appreciated!

    Thanks and best,

    Lior
    Attached Files

  • #2
    Hi Lior,

    Don't you have the possibility to check your samples with a Bioanalyzer or similar??
    I think it would be better...

    Comment


    • #3
      Hi Helena,

      Just yesterday did the bioanalyzer analysis. please see attached. This is after rRNA depletion by RiboZero and by mistake the analysis was done with Eukaryotic reference.

      The big 100-140 nt peak is tRNAs and sRNAs, right?

      Thanks and Good weekend,

      Lior
      Attached Files

      Comment


      • #4
        Yes, you are right, the peak should be miRNA, tRNA and so on.
        Good weekend!

        Comment


        • #5
          This paper has a nice method of assessing RNA quality not based on rRNA. Essentially they look for degradation from the 3' end based on RT-qPCR products from the same RNA using the same forward primer and various reverse primers. Assuming 3'-to-5' decay, a sample with extensive RNA decay will have less of the RT-qPCR products near the 3' end.

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