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  • pmiguel
    replied
    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

    Leave a comment:


  • RNAseqer
    replied
    Interesting comments about rRNA removal with ethanol precipitation. Any experience with removing rRNA while trying to recover only small RNA ?

    Leave a comment:


  • epibio
    replied
    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?

    Leave a comment:


  • pmiguel
    replied
    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.

    Leave a comment:


  • ETHANol
    replied
    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.

    Leave a comment:


  • pmiguel
    replied
    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

    Leave a comment:


  • ETHANol
    replied
    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

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  • pmiguel
    replied
    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

    Leave a comment:


  • epibio
    replied
    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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  • ag4lm4
    replied
    Thanks for the reply. I did not fragment depleted RNA before cDNA synthesis. Still wondering why could not anuthing on Agilent?

    Leave a comment:


  • epibio
    replied
    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.

    Leave a comment:


  • ag4lm4
    replied
    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!

    Leave a comment:


  • epibio
    replied
    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.

    Leave a comment:


  • Lucilia
    replied
    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?

    Leave a comment:


  • epibio
    replied
    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.

    Leave a comment:

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