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    • Troubleshooting

    Troubleshooting

    System inside GeneProof

    Troubleshooting guide is divided according to the main themes. This division will allow for electronization and implementation into the GeneProof Web site, including a continuous update. Information sources for the updates are OTRS, discussion with customers and others. Troubleshooting data are updated repeatedly.

    The troubleshooting logic copy the diagnostics process in standard molecular diagnostic laboratories. The general topic is always written in capital letters, which is then further expand according to the specific problem.

    GENERAL – issue with low or no fluorescence

    Low fluorescence

    Possible Cause Solution
    Degraded MasterMix Store at -20°C, thaw at 2 - 8°C until fully thawed. Vortex and short spin before use.
    Unverified cycler Use verified cycler. List of verified cyclers here.
    Incorrect consumables Use recommended consumables. List of consumables in device manual.
    Clear PCR strips on BioRad CFX96 Use white PCR strips on BioRad CFX96
    Incorrect transport medium Use TE buffer or Nuclease-free water.
    Air bubbles in PCR mix solution Spin PCR strip before PCR.
    Photosensitive MasterMix Minimize exposure of the MasterMix to light during handling to avoid loss of fluorescent signal intensity.

    Combination of parameters with unequal fluorescence on Rotor-Gene Q

    Place the positive control from the problematic PCR kit in well 1 and enable Auto-Gain Optimization, or run the problematic kit in a separate experiment.


    No fluorescence

    Possible cause Solution
    Incorrect PCR setup Contact support. Templates can be provided.
    Incorrect consumables Use recommended consumables. List of consumables in device manual.
    Incorrect transport medium Use TE buffer or Nuclease-free water.
    Photosensitive MasterMix Minimize exposure of the MasterMix to light during handling to avoid loss of fluorescent signal intensity.
    Degraded PCR reagents  Transport the PCR kit on dry ice.

    Combination of parameters with unequal fluorescence on Rotor-Gene Q

    Place the positive control from the problematic PCR kit in well 1 and enable Auto-Gain Optimization, or run the problematic kit in a separate experiment.

     

    All

    Was the answer helpful?

    YES NO Contact our technical support.

    SAMPLE – issue with low fluorescence

    Possible Cause Solution
    Insufficient extraction Use validated extraction.
    Unvalidated specimen Check list of validated specimen in IFU.
    Mishandling with sample Check sampling and sample storage in IFU.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    SAMPLE – issue with non-sigmoidal curves

    Possible Cause Solution
    Invalid curves caused by software Contact support.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    SAMPLE – false positive sample

    Possible Cause Solution
    Contamination Clean workspace.
    Cross-talk Contact support.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    INTERNAL CONTROL – fail of internal control

    Possible Cause Solution
    Incorrect volume Add 10 % of elution volume to sample before extraction.
    Degraded IC Store at -20 °C, thaw at 2 - 8 °C until fully thawed.
    Insufficient extraction Use validated extraction.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    INTERNAL CONTROL – fail of internal control + positive sample

    Possible cause Solution
    Possible competition Normal - still valid result.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    INTERNAL CONTROL – fail of internal control + negative sample

    Possible cause Solution
    Failed extraction Use validated extraction. Repeat whole analysis.
    PCR inhibition Repeat analysis of failed samples.
    PCR inhibition When working with GeneProof PathogenFree RNA Isolation Kit, eluate in 100 µl to dilute inhibitors.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    NEGATIVE CONTROL – positive signal

    Possible Cause Solution
    Contamination Clean workspace.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    POSITIVE CONTROL – negative signal

    Possible Cause Solution
    Degraded Positive Control Store at -20 °C, thaw at 2 - 8 °C no longer than 30 min. Vortex and short spin before use.
    Degraded MasterMix Store at -20 °C, thaw at 2 - 8 °C no longer than 30 min. Vortex and short spin before use.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    POSITIVE CONTROL – signal in HEX channel

    Possible Cause Solution
    Cross-talk Set threshold (TH) above curve in HEX.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    CALIBRATORS – issue with abnormal Ct

    Possible Cause Solution
    Incorrect pipetting Repeat PCR. Valid calibration curve values - R^2 ≥ 0,98; SLOPE between -3,8 to -3,1.
    All

    Was the answer helpful?

    YES NO Contact our technical support.

    SAMPLE – false negative sample

    Possible Cause Solution
    Incorrectly set threshold Set threshold lower/set threshold just above negative control
    All

    Was the answer helpful?

    YES NO Contact our technical support.

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