Contents - Index
Limit of Detection
(This feature is only available in GenEx Pro/Enterprise)
In many real life situations, particularly in diagnostics, it is important to know the sensitivity of a qPCR based analytical procedure. This can be assessed by determining the limit of detection experimentally, which is the number of copies that is necessary for reliable detection. This is done by analyzing standard samples of various concentrations. The concentration series should cover the expected limit of detection and each sample should be analyzed in replicates. The more replicates the higher the resolution, which will improve the accuracy of the estimation of the LOD. A minimum of six replicates at each concentration is recommended.
The data should include one classification column that holds the concentrations for each sample, and one that indicates the repeats so that samples that are repeats of eachother have the same value in the the repeats column.
Open the Regression tab among the analysis tabs in the top of the main window, and press the Limit of Detection button to load the analysis into the Control panel.
Select the classification column that holds the known concentrations of your standard sample dilution series in the Conc. column drop-down list. Also select the gene you want to use for calibration in the Gene drop-down list. The classification column that contains identifications of replicates is selected in the Repeat column drop-down list. Samples that are replicates should have the same unique value in the replicates column. Options are available to find confidence intervals with confidence level of 90%, 95%, 99% or Other. Fill in the CutOff cycle which ideally is higher than the cycle when the amplification of a single copy reaches threshold, but lower than the cycle when the amplification of primer-dimer products reaches threshold. The analysis is based on input of concentrations in linear scale. If you have concentrations on logarithmic scale, indicate this by ticking the Log scale check box before running the analysis.
Press the Run button to see the results. Based on the given cutoff cycle, samples are classified as either positive or negative. The result is presented as a cumulative distribution function (blue line) fitted to the fractions of positive samples at each concentration level (red dots) on a log-base 2 scale. The LOD cycle is presented in the Control panel. This is the concentration that can be detected with the given confidence level.