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  • onconaut
    replied
    Hello Bruce01,
    Ambion recommends to add the ERCC spikes prior to RNA selection. According to them it should also not make a difference whether you use poly-A selection vs. riboRNA depletion. However I have heard that poly-A selection introduces some bias as in that it does not capture all ERCC RNAs with the same efficacy. I have not done the experiment myself, nor have I seen the data, but that is one thing to keep in mind when you are doing your downstream analysis.
    We are using ERCC spikes-ins for all our library preps for now. Since we are working with archived RNA we have chosen riboRNA depletion over poly-A selection. We use Qbit for RNA quantification and have moved completely moved away from Nanodrop.

    Leave a comment:


  • bruce01
    replied
    Wondering if anyone tried these spike-ins from pre-polyA capture? I am hoping to use them with total RNA as I am using Truseq v2 kits. My issue is quantification of RNA concentration. Obviously the nanodrop and bio are pretty 'variable', would the Qubit be precise enough? It would seem that not accurately quantifying concentration would lead to levels of spike-in which could cause issues with normalisation which the product is meant to be ameliorating...

    Leave a comment:


  • onconaut
    replied
    This is extremely helpful. Thank you very much.

    Leave a comment:


  • HESmith
    replied
    A paper describing the use of ERCC controls for the Illumina platform can be found here.

    Leave a comment:


  • scbaker
    replied
    Illumina does know about them, or at least they used to. Illumina participated in their creation (mostly in the form of me attending the meetings and helping to define the controls) . Life has probably done a better job at incorporating them because they are sold by Ambion (owned by Life).

    They can be as useful as you want them to be - they're just a set of artificial RNAs at known concentrations. Depending on when and where you spike them in, you can control for all sorts of variables.

    It's too bad Illumina hasn't done more - the probes are built into all of the expression arrays and they've done quite a bit of testing with the NGS assays, but I guess they don't have any official support. You might have better luck talking to someone in the bioinformatics department. They're the ones in R&D who spent the most time working on them and how best to do the analysis for NGS samples.

    Good luck!

    Leave a comment:


  • onconaut
    started a topic ERCC spike in controls for illumina?

    ERCC spike in controls for illumina?

    Hello Seqers,
    We have a illumina technician installing our HiSeq2000 as I write and I am trying to make some final improvements on my library constructions especially in the area of quality control.
    I saw that life tech has ERCC spike in controls pretty well incorporated into their protocols and pipeline. When I was looking for this in combination with illumina protocols I found nothing. When I called illumina tech support no one had ever heard of ERCC.

    Does anyone have experience with the ERCC controls?
    Do they really add that much more information in terms of helping helping me interpret my data?
    Why does illumina not use them/support them/know about them?

    Thanks a lot for your insights,
    O.

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