The experiment has been carefully planned, set up and run. The run is finished and now it is time to analyze the data. What to look at first? Do we really know what the analysis settings do? Does it matter? The most common place to start preliminary analysis of a real-time PCR run is using the Amplification view that allows adjustment of the baseline and threshold values. The default view for most software plots cycle number on the x axis versus the logarithm of the ARn on the y axis. Figure 2.3 shows a flowchart with suggested steps of parameters to check and in what order. The steps in the flowchart are meant to be a guideline and it may be necessary to adjust some parameters, such as the threshold, more than once. Some instruments require that a baseline and threshold be accepted before further manipulations of the software can be executed, but all instruments allow the user to change the baseline and threshold and reanalyze at will. All analysis scenarios presented in this chapter assume that the instrument is working properly.
Effect of incorrect dye layer settings. A. Amplification view showing the incorrect dye layer (TAMRA). B. Amplification view showing the incorrect dye layer (SYBR®). C. Amplification view showing the correct dye layer (FAM). D. Curves showing the relationship among spectra for the dyes FAM, SYBR® (Green I) and TAMRA. Arrow at 'a' identifies Ct of the same amplification curve under different analysis conditions.
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