What is the Quality Threshold score, how is it calculated and why does it fail my data?
The Quality Threshold in the Biomark system analysis software is a qualitative tool designed by Fluidigm to measure the "quality" of each amplification curve. Each amplification curve is compared to an ideal exponential curve and given a Quality Score between 0 and 1, where 0 is a flat line and 1 is a perfect sigmoid.
Factors taken into account include the linearity of the baseline, the delta Rn of the final product or signal that is produced, the slope of the amplification plot and the return to linearity after exponential growth.
An arbitrary cutoff value of 0.65 is the default setting for the Quality Threshold in the Real-Time PCR Analysis software. This cutoff is user-adjustable and can be increased for more stringency or decreased for less. Also, passed or failed amplification curves can be manually changed. The Quality Score for each Ct value serves as a flag and does not remove that value from the dataset.
A failed Quality Score will be removed from two other types of calculations: the Ct threshold calculation and a reference normalization calculation. For example, if the reference gene Ct has a failed Quality Score, it will not be used in a dCt calculation.