Troubleshooting a real-time PCR assay is an essential part of assay optimization and validation. Researchers can significantly reduce erroneous data and downtime by going through this checklist:
- Ensure that primer-dimers are not contributing to signal or poor efficiency
- Take steps to minimize PCR inhibition and poor reaction efficiency, for example by re-purifying the template or using an appropriate magnesium concentration
- Confirm appropriate software and instrument analysis settings
- Make standard curve validation the final step in the reaction assessment process
- Understand how to address efficiencies below 90% versus those above 110%
- Mitigate the sources of no amplification
- Identify factors that most seriously affect data accuracy, particularly amplification curve baseline linearity, baseline range settings, threshold, and reference dyes
The culprits of unsuccessful real-time PCR assays are many and varied. Identifying the root causes of analysis issues is the first step in successful troubleshooting.
In addition to troubleshooting tips, our “Real-time PCR handbook” is the definitive source for all things related to real-time PCR, including:
- Basics of real-time PCR
- Experimental design
- Plate preparation
- Data analysis
- Digital PCR
Download your FREE copy of the “Real-time PCR handbook” today!