i would like to analyze a qRT PCR (SYBR green) with Real-time PCR Miner Version 4.0. Therefore I need the gene average efficiency, but i have no idea where to find it. The instructions say it is in the raw data, but i dont have it there. I use Applied Biosystems Step One plus. Did anyone maybe solve this problem already?
Hello Nadia,
The PCR amplification efficiency can be either defined as percentage (from 0 to 1) or as time of PCR product increase per cycle (from 1 to 2).
The efficiency-calibrated model is a more generalized ΔΔCt model. Ct number is first plotted against cDNA input (or logarithm cDNA input), and the slope of the plot is calculated to determine the amplification efficiency (E). ΔCt for each gene (target or reference) is then calculated by subtracting the Ct number of target sample from that of control sample.
How you can appreciate, if you want to do this using the software of your thermocycler, you must include one reference rather than a sample (specifying it in the thermocycler software). Once you specify all this, you can export your data for further analysis. If you are a customer of an Applied Biosystems Step One plus, i think that you can use the cloud "Applied Biosystems® qPCR Analysis Modules" (https://www.thermofisher.com/es/es/home/technical-resources/software-downloads/ab-secondary-data-analysis-modules/applied-biosystems-analysis-modules-for-qpcr.html)
I have attached one documents that could be very useful for you.
Dear félix, thank you very much for the detailed answer and the documents!
- Badges
- Science topic