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  • Agencourt RNAclean XP bead purification troubleshooting

    I recently prepared some low-input rna-seq libraries using NuGEN's Ovation RNA-seq system for model organisms (mouse). After the final step I ran the libraries on a Bioanalyzer and saw that they looked good but had a large population of adaptor dimers present.

    Here is a representative image of one of my sample libraries, with a large adaptor dimer peak at ~125bp



    I wanted to get rid of the dimers before sequencing, so I did an extra bead purification step using a fresh tube of Ampure RNAclean XP beads that was supplied with the kit (I had previously used the same kind of beads several times during the procedure). I used a bead:sample ratio of 0.8:1.0 in order to remove fragments >200bp to get rid of the adaptor dimers. I followed the protocol exactly including fresh EtOH, optimal drying time, etc. I re-ran the samples on the Bioanalyzer after the purification and I got some very strange results that I'm having trouble interpreting. I have run the post-purification samples twice on the Bioanalyzer with identical results.

    Here is a sample of the same library as above, after a bead purification. Note high MW DNA that was previously not present in the sample, as well as a complete lack of most of the DNA that I had seen before. Several of the samples had no detectable DNA other than the upper and lower DNA marker.



    Has anyone seen results like these? I'm not sure what could have gone wrong with the bead purification unless I somehow got a bad batch of beads. That still wouldn't explain the new presence of high MW DNA that appeared after purification. I'm hoping it's a problem with the Bioanalyzer but I have no idea what it would be and I haven't found anything during my search of literature and online.

    Any help is greatly appreciated, thanks!

  • #2
    Bioanalyzer issues can be checked by looking at ladder run. If ladder looks OK then it is less likely to be instrument related. One possibility is carryover of beads during collection of purified DNA. This can be fixed by putting eluted samples back on magnet and recollecting. By the way, if the top sample is final RNA library (not SPIA amplified cDNA) it looks odd because of distinct bands.

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    • #3
      Originally posted by nucacidhunter View Post
      Bioanalyzer issues can be checked by looking at ladder run. If ladder looks OK then it is less likely to be instrument related. One possibility is carryover of beads during collection of purified DNA. This can be fixed by putting eluted samples back on magnet and recollecting. By the way, if the top sample is final RNA library (not SPIA amplified cDNA) it looks odd because of distinct bands.
      The ladder was fine, there were strong peaks for all of the standards. Thanks for the magnet suggestion, if I do anything more with the samples I'll keep them on a magnet for a while before pipetting them.

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      • #4
        Did you find the reason for this? I got a very similar result with high MW fragments in some of my libraries, as shown in the second figure. However, one sample in the batch gave "normal" curve so I guess the protocol and reagents should be ok. I tried an additional incubation on magnet an also made new libraries (Nextera XT) and repeated the ampure purification wit a new batch. Still got similar results. Are there any other suggestions that may cause the presence of high MW fragments in some of the samples and do you think those will interfere with Miseq sequencing?

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