For your convenience, we have created a file template using Microsoft® Excel that enables the optimization of algorithm constants with data collected using  Qubit® Raw mode  (right click, save target as, save to desktop) This file includes space for triplicate samples of eight different analyte concentrations. Depending on your needs, you may wish to use more or fewer sample concentrations and additional replicates. Accuracy and reproducibility will generally improve as the amount of data used to refine the algorithm constants increases. You may also wish to collect data from multiple independent experiments to assess the reliability of your new assay.

The data template provided calculates concentration values for each sample based on the fluorescence reading obtained on the Qubit® 2.0 Fluorometer (or other fluorescence-based device) and the constants entered into the algorithm at the top of the page (the fields in blue). These concentration values are reported in the section “Calculated Outputs.” By default, the template defines the highest concentration as Standard 2. However, you may adjust the value of Standard 2 according to the accuracy of output, the desired dynamic range requirements for your assay, or both. The template lists the deviations from the expected concentrations for each sample under the heading “% Deviation” and all of these results are averaged and summarized at the right-hand portion of the open file. From this summary, you will be able to see that the average deviation and accuracy of your samples is based on the constants entered at the top of the file. You may wish to adjust the constants until you have obtained results with which you are comfortable.

Note: Data collected using a traditional fluorescence microplate reader (or other fluorescence-based device) may also be analyzed using this template. However, some assays may exhibit different standard curve shapes when using the Qubit® 2.0 Fluorometer, and for this reason, we recommend confirming performance on the Qubit® instrument using Qubit® Raw mode  prior to uploading a new assay using MyQubit firmware.