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  • Ribo-Zero

    Hello
    I am struggling with mRNA extraction from frozen tissues.
    Unlike fresh tissue, frozen tissue includes lots of degraded rRNAs.
    Although FAS from SOLiD recommended to use poly-A capture, I'd like to prepare mRNA from total RNA by removing rRNAs.
    I was told that Ribo-Zero was much better than Ribominus to remove rRNA.
    Anyone has experience with Ribo-Zero?

  • #2
    Ribo-Zero kits should give you very good rRNA removal from degraded RNA samples; see this blog post for an example.
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    • #3
      Seemed to work well for us on some partially degraded sheep RNA. Not sure how well it depleted the rRNA yet -- the run is going now.

      We had a little trouble with low yield initially but turned out it was low efficiency of precipitation. When we switched to Zymo columns for the final RNA concentration step our yields stabilized.

      --
      Phillip

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      • #4
        Originally posted by epibio View Post
        Ribo-Zero kits should give you very good rRNA removal from degraded RNA samples; see this blog post for an example.
        looks good

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        • #5
          I have been using RiboZero with both normal and low input on all types of RNA including highly degraded FFPE with great results. Definitely better than RiboMinus.

          We generally do whole transcriptome and have been happy with the results.

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          • #6
            Is it because ethanol precipitation tends to loose small RNA while Zymo promises to keep everything above 17?

            ...but at the end of the day, most sequencing protocol will remove small fragments anyway. Say ILMN PE seq. will remove everything under 150 or 200 or longer depending on circumstances?

            Originally posted by pmiguel View Post
            Seemed to work well for us on some partially degraded sheep RNA. Not sure how well it depleted the rRNA yet -- the run is going now.

            We had a little trouble with low yield initially but turned out it was low efficiency of precipitation. When we switched to Zymo columns for the final RNA concentration step our yields stabilized.

            --
            Phillip

            Comment


            • #7
              No, I think it is because with ethanol precipitation you tend to lose all your RNA, especially when attempting to precipitate low concentration solutions. Or at least this is not an uncommon outcome.

              --
              Phillip

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              • #8
                Quick question. How does ribo-zero target rRNA? I do not want to lose mRNA that is bound in ribosome complex.

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                • #9
                  Are you purifying polysomes? The kits are designed to work with purified total RNA, so there shouldn't be any mRNA bound in ribosomes. The rRNA is removed by solution-based hybridization capture.
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                  • #10
                    ribo-zero plants

                    Originally posted by pmiguel View Post
                    Seemed to work well for us on some partially degraded sheep RNA. Not sure how well it depleted the rRNA yet -- the run is going now.

                    We had a little trouble with low yield initially but turned out it was low efficiency of precipitation. When we switched to Zymo columns for the final RNA concentration step our yields stabilized.

                    --
                    Phillip
                    Do this kit work well for plant material?

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                    • #11
                      Yes, we have two types of plant Ribo-Zero kits (for RNA from seeds or roots/leaves). We will be presenting a poster on plant RNA-Seq at PAG XX next week.
                      Last edited by epibio; 01-11-2012, 08:11 AM.
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                      • #12
                        What the size of the cDNA should be if RiboZero rRNA depleted sample and hexamers are used? I cannot see anything on the High Sensititvy bioanalyzer and at the same time nano drop gives about 20ng/ul. Is it because cDNA has a high molecular weight(longer that 10kb)? Thanks!

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                        • #13
                          It depends on your library prep method (for example, fragmentation conditions). With our ScriptSeq library prep, we typically get a range of 200-1,000 bp with a peak around 350 bp. You would not expect 10 kb cDNA with a random-primed method.
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                          • #14
                            Thanks for the reply. I did not fragment depleted RNA before cDNA synthesis. Still wondering why could not anuthing on Agilent?

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                            • #15
                              Can your provide more information about: i) the source and quality of the total RNA; ii) how much rRNA-depleted RNA you used as input for library prep; iii) the library prep method, including clean-up steps?
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