Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Small RNA library with 200bp extra peak

    I just looked at our new small RNA library submitted by a respected lab on the Bioanalyzer. The small RNA library should be about 178, and it's there, but most of the samples have a broader but distinct peak at 200bp. I've posted a link to an example below. Any ideas what this could be and even more, how to eliminate it?

    Thanks.
    Pete

  • #2
    That to me looks like over-amplification, we have seen 'bulged' products that run at 200-250bp. See page 9 of this protocol from the Mello lab (http://www.lsi.umich.edu/files/SmallRNACloning.pdf)

    What kit was used to prepare the libraries? 178 is big for miRNAs unless you are using QIAseq.

    Cheers,
    sergio

    Comment


    • #3
      You're right, that does resemble over amplification. I was going to run the samples on a denaturing gel but that protocol says not to. Can it be fixed? I think the protocol was mostly custom. I'm not someone who makes small RNA libraries yet, so I can't really comment on what was custom and what may have come from a kit.
      Last edited by hoytpr; 02-12-2018, 04:51 PM.

      Comment


      • #4
        Bulged products are not a problem for sequencing, you only see them with gels or bioanalyzer. For sequencing you denature the libraries anyways. So there is nothing to fix, just that they probably amplified the libraries for too many cycles.

        Comment


        • #5
          Originally posted by sbarberan View Post
          Bulged products are not a problem for sequencing, you only see them with gels or bioanalyzer. For sequencing you denature the libraries anyways. So there is nothing to fix, just that they probably amplified the libraries for too many cycles.
          So, you'd have to quantify by QPCR then, as fluorescence or Bioanalyzers won't tell you the number of correctly formatted sequences with adapters?

          Comment


          • #6
            You are probably right that qPCR will be the only way to quantify with 100% accuracy, but generally we do not use qPCR and even with libraries with bulged products we manage to quantify only with Qubit and Bioanalyzer and get great pooling.

            Alternatively you can also try to denature the sample before loading into the Bioanalyzer.

            Comment

            Latest Articles

            Collapse

            • seqadmin
              Strategies for Sequencing Challenging Samples
              by seqadmin


              Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
              03-22-2024, 06:39 AM
            • seqadmin
              Techniques and Challenges in Conservation Genomics
              by seqadmin



              The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

              Avian Conservation
              Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
              03-08-2024, 10:41 AM

            ad_right_rmr

            Collapse

            News

            Collapse

            Topics Statistics Last Post
            Started by seqadmin, Yesterday, 06:37 PM
            0 responses
            8 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            8 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-22-2024, 10:03 AM
            0 responses
            49 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-21-2024, 07:32 AM
            0 responses
            66 views
            0 likes
            Last Post seqadmin  
            Working...
            X