Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Wacky BioAnalyzer Results

    Hi All

    Has anybody ever seen anything like these?
    I'm asking this question in as many forums as possible since nobody around me seems to have any clue what is going on - even the techs at Agilent say they've never seen traces like these.

    They are total RNA isolations. I have troubleshot this issue several times, thought that I'd solved the problem, only to have it crop back up again and be solved again.

    Please assume that i am following the Agilent manual fairly closely at this point and have even added steps suggested by Agilent techs from my previous troubleshooting sessions.

    Earlier solutions that seemed to have fixed the problem before include extra time to equilibrate all reagents to room temp, vortexing the prepped chip at 2000 rather than 2400, and allowing extra time for the electrodes to fully dry after RNase-free water wash. The same samples that gave me problems, would then read just fine once each of these changes in protocol were made. I have also tested for vibration from a nearby centrifuge and shearing of the gel by high speed centrifugation.

    Several other people, from several other labs are using this same machine [often times in between 2 of my runs] and nobody is getting anything even close to this. The issue is absolutely something I'm doing wrong with my prep.

    The attached photos are recent, from one of four runs using the same samples. No sample gave the same results twice in any of the four runs. Clearly the samples are not the problem.

    HELP!

    Thanks
    Attached Files

  • #2
    How are you isolating your RNA? We've seen traces like this, when the purity of the RNA isn't good.

    Comment


    • #3
      Originally posted by ZWB View Post
      How are you isolating your RNA? We've seen traces like this, when the purity of the RNA isn't good.
      Isolating with a modified RNeasy protocol. Have optimized for purity. Also, samples that give these results will then give very good reads - even high RIN values - once I 'fixed' the issue in the past. I'm quite sure at this point that it isn't the samples.

      I've tested for possible contaminants in my Agilent reagents in the past, but nothing came of that either.

      Comment


      • #4
        ... also, check out the ladder read - even those come out all wonky and never in the same way between chip runs

        Comment


        • #5
          Have you run one with just buffer and ladder and no sample? When I have seen oddities like this it is usually due to high carry over salt into my samples.

          Comment


          • #6
            My guess is you ladder is degraded. It happened to ours and the images were totally whacky. I don't remember exactly what they looked like but it was probably something like what you see where you just look at it go WTF?

            I think the software just goes nuts when it can't find the correct ladder sizes or at least that was my explanation. We got a new kit and my RNA looked great again. The kits start to go bad even before the expiration dates.
            --------------
            Ethan

            Comment


            • #7
              Are you denaturing your ladder and samples for a few minutes before loading on the chip?

              Comment


              • #8
                Hi guys

                I heat denature everything before i run. The ladder was changed up in the middle of these attempts. I've added another run, showing the exact same 12 samples, with the new ladder. Same thing.

                Also, notice that the same sample never gives the same result twice - these 'reads' are almost surely the machine reacting to something other than the actual samples

                In the past I've tried switching over to a new lot of chips and changing over the entire reagent set. To no avail.
                Attached Files

                Comment


                • #9
                  Do you scrub the electrodes regularly?

                  Comment


                  • #10
                    Oh, also, i should mention that in the past, when i have 'troubleshot' this issue, the same samples that were giving me problems would then give often time excellent results. I've also added positive controls in the past which either gave bad results if the overall read was bad, or good if the overall read was good.

                    Comment


                    • #11
                      I decom the electrodes with RNase-Away and then RNase-free water before starting everytime

                      Comment


                      • #12
                        In addition to that they need regular scrubbing. We scrub ours with a sterile toothbrush first with MilliQ water, then isoproponal, and again with MilliQ. you then need to let them dry out very well (preferably over a long weekend).

                        We do it weekly, but our BioAs are used at 100% capacity if yours are used less this should be done every few weeks or maybe monthly.

                        Comment


                        • #13
                          Someone did just do the routine cleaning last week, but again others are not getting anything like this - of the five runs I've done with these same 12 samples, all giving crap results, someone slipped in and ran a DNA chip in the hour i gave them between 2 runs and got perfectly fine results. :-|

                          Comment


                          • #14
                            I would repurify the samples, sounds like maybe you have a contaminant in the sample screwing with the run.

                            Comment

                            Latest Articles

                            Collapse

                            • seqadmin
                              Exploring the Dynamics of the Tumor Microenvironment
                              by seqadmin




                              The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
                              07-08-2024, 03:19 PM
                            • seqadmin
                              Exploring Human Diversity Through Large-Scale Omics
                              by seqadmin


                              In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
                              06-25-2024, 06:43 AM

                            ad_right_rmr

                            Collapse

                            News

                            Collapse

                            Topics Statistics Last Post
                            Started by seqadmin, 07-19-2024, 07:20 AM
                            0 responses
                            26 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 07-16-2024, 05:49 AM
                            0 responses
                            41 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 07-15-2024, 06:53 AM
                            0 responses
                            46 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 07-10-2024, 07:30 AM
                            0 responses
                            42 views
                            0 likes
                            Last Post seqadmin  
                            Working...
                            X