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  • Amilification kit to low amount of Total RNA

    Hi,
    i am planning a seqencing to ~5ng of total RNA by using HiSeq2000.
    i wonder which of the amplification kit will be better to use.

    SMARTer Ultra Low RNA Kit (Clontech)
    or
    Ovation RNA-seq v2 (Nugen)

    Does anyone have any an experience with one of them?
    Recommendations?

    Thanks,
    pap

  • #2
    For me the Clontech SMARTer kit worked beautifully. Used 100pg total RNA and generated nice Bioanalyser profiles. Also titrated down from 50 to 1 cell and got good products with all of them. You do have to optimise the cycle number, of course, and at the single cell level (which is way below the spec of the kit) there is some inevitable variation, but its pretty impressive.
    Tried the Nugen kit and didnt really get on with it, but maybe that was just me.

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    • #3
      I've tried both protocols and for me the best one is the NuGEN Ovation kit v2. You need only 500pg RNA to start and in few hours you are able to amplify it. I think it is also more user-friendly compared to Smarter.

      Comment


      • #4
        SMARTer all the way for us.
        We had some strange mapping results from the NuGen kit with lots of intronic mappings. In the end we ignored them and the results did correlate well with array data (from the same total RNA), but it's a waste of sequencing capacity. I think there are other posts on here where people mention they had similar problems with intronic reads. I suspect the kit may be very sensitive to any (DNA) contamination due to the use of random priming.

        Techniques and protocol discussions on sample preparation, library generation, methods and ideas


        The SMARTer kit on the other hand produced very clean data, even from a single cell - so it's very sensitive. The downsides are the protocol isn't as user friendly as NuGen and it's much more expensive. It also requires a bit of expertise to optimise the library prep post amplification. There was also greater disparity between the array data and the SMARTer data, but we expected this from single cell work.

        Comment

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