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How to Measure PCR Efficiency of an Assay -- Ask TaqMan®: Ep. 4

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Published on Jun 26, 2012

Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding of PCR efficiency and how to properly measure it in your assays. To check the efficiency of an assay, you need to 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.
Happily, with all newer AB instrument software, the efficiency calculation is actually done for you. Just go to Results  Standard Curve, and there it is. Now, here's the thing: I see a lot of cases where people make mistakes preparing their dilution curves, and as a result, calculate incorrect efficiencies. So what I want to do is give you TEN . . . suggestions on how to improve the accuracy of your dilution curves.
#10: Use well-calibrated pipettemen
#9: Serially dilute standard material
#8: For AQ, always use an appropriate standard material
#7: Mix standards thoroughly after each dilution
#6: Pipette higher volumes for better precision
#5: Run ≥ triplicates of each dilution point in real-time
#4: Use minimum of 5 dilution points, 10-fold dilutions
#3: After run, check R2 / look for outliers; omit.
#2: If curve has more than 1 or 2 outliers, or if the spacing between points is irregular, repeat curve
#1: Have more than one person run a curve the first time to check validity
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." That document is available for download on the Life Technologies website.

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