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  • ribosomal RNA depletion

    Hello,
    I'm using Epicentre Ribo-Zero kit for removal of rRNA from Drosophila. I checked RNA on Agilent, before and after depletion, and I observed a picture that seems strange for me (attached). A lot of small RNAs that do not seem to be present in the initial sample and nothing that looks like mRNA peak (it should be about 1000-2000 nt, I guess).
    What is your opinion, is it normal?
    Attached Files

  • #2
    Hi,

    Drosophila is not the best substrate for Ribo-Zero, but it does work (see attached data). Not all rRNA species are readily removed, but if you do a Google Scholar search under "Ribo-Zero" and "Drosophila" there are at least one or two papers that cite this combination.

    Here's an example:

    Kumar et al. BMC Research Notes 2012, 5:230

    I hope this is helpful.

    Olaf
    Attached Files

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    • #3
      Hello MLog,

      How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


      Thanks for the sharing,
      Wei

      Comment


      • #4
        Originally posted by weigrc View Post
        Hello MLog,

        How is the alignment result for above samples, I mean the % of sequencing reads mapped to rRNA?


        Thanks for the sharing,
        Wei
        Hi Wei,
        not as good as I wish, actually - about 30-40%. Next time I did double depletion and it worked much better, with ~ 7% mapped ro rRNA.

        Comment


        • #5
          Hello MLog,

          For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

          What is your input amount of the total RNA?

          Thanks,
          Wei

          Comment


          • #6
            Originally posted by weigrc View Post
            Hello MLog,

            For the 1st depletion, do you think less input of the total RNA can help to reduce the 30-40% mapped rRNA??

            What is your input amount of the total RNA?

            Thanks,
            Wei
            I usually take 1-3 micrograms of total RNA. With human and bacterial RNA it worked well - less than 1% of reads mapped to rRNA. Probably for Drosophila it's worth trying to take less.

            Comment

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