Encountering a failed control in a qPCR run can be frustrating, but it's important to understand the reasons behind it and the necessary actions to take. For more information on the different types of controls, read the following article: What type of controls are present in qPCR?. Here, we'll explore why your positive, internal, and negative controls might fail and how to proceed.
Why did my positive control fail?
Several factors could lead to a positive control's failure:
- Ct Value Outside Expected Range: The Ct value for a positive control should typically fall between 15 and 25. Values outside this range may indicate an issue.
- Incomplete Rehydration of Freeze-Dried Pellet: Ensure that the freeze-dried pellet is fully rehydrated by waiting the complete 5 minutes as instructed.
- Pellet Dislodgment or Misplacement: Verify that the pellet is present in the assay tube before rehydration.
- Degraded Enzyme: Using enzyme that has degraded after initial rehydration can lead to failure. Consider using a new positive control vial.
- Expired or Improperly Stored Reagents: Reagents that are past their expiry date or have been exposed to extreme temperatures may degrade prematurely.
Should I reject my results if my positive control fails?
Upon encountering a failed positive control, it's crucial to check the Ct value. While a failed positive control is concerning, a significant Ct value (e.g., less than 35) could still indicate a successful run, despite the control's failure.
For example, the positive control for Legionella Pneumophila should be between 15-25 (for the software to call it a pass). A significant Ct value of less than 35 would be considered a good run despite the result indicating a positive control= fail.
Example: Well B4 - The Positive Control failed, but the CT value is 26.83
Why did my internal control fail?
The internal amplification control can fail if the target DNA is high or if inhibitors are present in the extracted sample.
Example: Well A5 Target Amplification (Green) but no internal control amplification (Orange)
Interpretation: Positive result
Example: Well A5 No Internal Control Amplification- possible inhibition
Inhibition will be suspected due if there is no internal control or target amplification. If there is no internal control or target amplification, it would be recommended to perform a 1:10 dilution of the eluted DNA to dilute out qPCR inhibitors.
Any dilution performed would need to be noted on the sample for later analysis. It can be valuable to run samples again when strange results are observed, in this example, perhaps next to a diluted replicate to rule out inhibition.
Why did my Negative Control Fail?
A failed negative control usually indicates contamination. Here’s what to do:
- Check for Contamination: Identify any potential sources of contamination.
- Use New Supplies: Replace any potentially contaminated reagents or consumables.
- Clean Area Well: Ensure the workspace is thoroughly cleaned and free of contaminants.
- Rerun the Experiment: Perform the run again with the new supplies in a clean environment.