Screen shot of a prototypic Java program that automates LRE quantification. Made up of several panels that bring together all of the components of LRE quantification, this program allows the interrelationships of each component and their impact on the calculated target quantity to be examined. 1. Lists the FC dataset. 2. The LRE plot which allows manual adjustment to the LRE window (denoted by circles). 3. The F0 plot from which target quantity is derived by averaging the F0 values within the LRE window (denoted by red circles). 4. Numerical summary with the red lettering denoting the FC readings encompassed by the LRE window. 5. The FC plot which compares actual (dots) to predicted (circles) FC, with the red circles denoting the FC readings encompassed by the LRE window. 6. Ct determination based upon user-selected fluorescence threshold based on linear regression analysis of the log-linear region of the amplification profile (implementation details available from the corresponding author upon request). 7. Target quantification where the number of target molecules is calculated based upon four parameters: average F0 taken from 3, whether the target is double or single stranded, the amplicon size, and the optical calibration factor (OCF) (see text for details). Conversion of Ct to N0 via F0 is also included, which allows the quantitative differences between LRE- and Ct-based quantification to be assessed. 8. Buttons provide the ability to paste a FC dataset from the clipboard and to initiate automated analysis ("Paste & Analyze"), or to repeat the automated analysis on the current FC dataset ("Reanalyze"), or to reset the FC dataset to the default provided with the program ("Reset"). 9. Provides a summary presented as a dialog box that that can be copied to clipboard and pasted into Excel.
Rutledge and Stewart BMC Biotechnology 2008 8:47 doi:10.1186/1472-6750-8-47