Whether you’re a beginner or a seasoned veteran in the lab, you probably have questions about real-time PCR. The Life Technologies staff of scientists are participating in a new project called, “Ask TaqMan®.” Customers ask questions and our scientists answer them in easy how-to videos about best practices in real-time PCR.
Here is a Tip from the Ask Taqman Series:
To check the efficiency of an assay, run a template dilution curve in real-time, and then look at the resulting slope of that curve.
That slope value -- assuming you prepare the curve properly -- tells you the efficiency. If the slope is a -3.3, then the assay is amplifying at or very near 100% -- exactly what we want in real-time PCR. But if that value is more negative -- say, -3.7 -- then the efficiency will be less than 100%. There's actually a formula into which you can plug this slope and get your numerical efficiency.
With all newer AB instrument software, the efficiency calculation is actually done for you. Just go to Results -> Standard Curve, and there it is. We see a lot of cases where people make mistakes preparing their dilution curves, and as a result, calculate incorrect efficiencies.
Here are Ten Suggestions to Improve the Accuracy of Your Dilution Curves
1. Have more than one person run a curve the first time to check validity
2. If curve has more than 1 or 2 outliers, or if the spacing between points is irregular, repeat curve
3. After run, check R2 / look for outliers; omit.
4. Use minimum of 5 dilution points, 10-fold dilutions
5. Run ≥ triplicates of each dilution point in real-time
6. Pipette higher volumes for better precision
7. Mix standards thoroughly after each dilution
8: For AQ, always use an appropriate standard material
9: Serially dilute standard material
10: Use well-calibrated pipettemen
One good thing to note is that all the TaqMan® Assays Life Technologies sells are designed to work at or near 100% efficiency. In fact, we tested thousands of these and published the results in an application note entitled "Amplification Efficiency of TaqMan® Gene Expression Assays."