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  • So, I just ran two different assays on the same Bioanalyzer Chip.

    This was a complete act of desperation but after accidentally running my Agilent RNA Pico Chip on the mRNA assay instead of the Total RNA Assay, and having just used the last ladder aliquot, I left the chip in there and just ran the Total RNA Assay. To my surprise it worked! See attached comparison.

    This is what I did:
    1. Loaded chip with precious samples
    2. Ran RNA assay.
    3. Realised I actually ran the mRNA Assay instead of the Total RNA Assay
    4. Realised I had no more ladder.
    5. Almost had a heart attack
    6. Let chip finish.
    7. Changed assay to Total RNA Assay and pressed start.
    8. Stalk machine in case it completely broke.
    9. Prayed for it to work

    It worked!

    This seems really tempting to run both an mRNA and Total RNA assay on my samples in future if I don't have to waste reagents. The drawback would be time, but I do Ribo Depletion on my samples at the moment and my usual workflow has been to extract RNA --> run Total RNA assay--> Ribo Deplete --> run mRNA Assay. For the sake of showing the efficiency of my depletion it might be nice to extract RNA --> Run Total + mRNA Assay ---> Ribo Deplete ---> Run Total and mRNA Assay, maybe even just for a couple of samples.

    My question is do you think this might damage the machine? It makes sense for the chip to hold extra reagents than is needed for one run.

    Side note: The samples I ran on this chip were actually cDNA samples that were denatured and snap cooled to test the presence of concactamers in some weird TotalScript libraries that had a second bump.
    Attached Files

  • #2
    Originally posted by SS00 View Post
    This was a complete act of desperation but after accidentally running my Agilent RNA Pico Chip on the mRNA assay instead of the Total RNA Assay, and having just used the last ladder aliquot, I left the chip in there and just ran the Total RNA Assay. To my surprise it worked! See attached comparison.

    This is what I did:
    1. Loaded chip with precious samples
    2. Ran RNA assay.
    3. Realised I actually ran the mRNA Assay instead of the Total RNA Assay
    4. Realised I had no more ladder.
    5. Almost had a heart attack
    6. Let chip finish.
    7. Changed assay to Total RNA Assay and pressed start.
    8. Stalk machine in case it completely broke.
    9. Prayed for it to work

    It worked!

    This seems really tempting to run both an mRNA and Total RNA assay on my samples in future if I don't have to waste reagents. The drawback would be time, but I do Ribo Depletion on my samples at the moment and my usual workflow has been to extract RNA --> run Total RNA assay--> Ribo Deplete --> run mRNA Assay. For the sake of showing the efficiency of my depletion it might be nice to extract RNA --> Run Total + mRNA Assay ---> Ribo Deplete ---> Run Total and mRNA Assay, maybe even just for a couple of samples.

    My question is do you think this might damage the machine? It makes sense for the chip to hold extra reagents than is needed for one run.

    Side note: The samples I ran on this chip were actually cDNA samples that were denatured and snap cooled to test the presence of concactamers in some weird TotalScript libraries that had a second bump.
    Not sure what you got out of total RNA run assay that you could not get out of the mRNA assay...

    My understanding is that only a portion of the sample actually migrates into the chip during "electro-injection". (Not sure that is the correct term.) I think a technical rep for Affymetrix told us it was possible to pippette sample from the loading well of a previously run chip into a new chip.

    Anyway, I don't think you would be able to trust concentration estimates of your second chip runs as there could be differential depletion of the the spike-in standard vs. your sample during injection.

    That said, it is still an interesting result. Thank you for sharing it with us.

    --
    Phillip

    Comment


    • #3
      I believe the only difference between the Pico mRNA assay and Pico total RNA assay is whether the software reports a rRNA contamination percentage or RIN value. In my opinion both of these calls involve a little bit of Agilent voodoo, and total RNA and mRNA quality are best assessed by considering the actual trace and using the reported value as a guide.

      However, I believe that Pico vs Nano protocols actually involve differences in the run, so it is very useful to know that a chip can be run again if the wrong assay is accidentally chosen the first time. Thanks for sharing! Bedankt!

      Comment


      • #4
        On a related note, you can also mix and match reagents and chips. As long as the protocol you run on the machine matches the reagents you should be gold. You need to load the reagents according to your assay.
        This is really handy as we often have more reagents than chips. Helpfully, Agilent sell the reagents only, but not the chips only.
        So far we've run DNA HS on a standard DNA chip and a DNA 1000 assay on an RNA Nanochip without problems.

        Comment


        • #5
          I used the wrong assay.
          It was set to prokaryote total RNA assay. I did not change the assay to eukaryote total RNA assay when checked quality for fish RNA.
          Does anybody know how to re=analyze the data? Thanks.

          Tom

          Comment


          • #6
            The traces should show the profile or your RNA no matter what assay you chose for the run. I recommend posting the profiles here in a new thread if you are unsure about your RNA quality, I am sure at least a few people can help.

            Originally posted by Tom2013 View Post
            I used the wrong assay.
            It was set to prokaryote total RNA assay. I did not change the assay to eukaryote total RNA assay when checked quality for fish RNA.
            Does anybody know how to re=analyze the data? Thanks.

            Tom

            Comment


            • #7
              Can a RIN be calculated from mRNA assay results?

              I am reawakening this thread because we ran mRNA instead of total RNA assays on the bioanalyzer, and are now left with the unhelpful rRNA contamination value, whereas what we actually do want is a RIN. Is there a way to (non-manually) get the RIN from the RNA traces after running mRNA assays? The traces themselves look fine, consistent with what the other respondents to this thread have said. Any advice would be really helpful. Thanks.

              Comment

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