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Perform your serial dilutions with a 1:10 dilution, starting at 1000 copies per reaction (this is an input level that is very likely to result in target detection) down to 1 copy per reaction (this is an input level that is likely to be below the LoD).Ģ.For a qPCR assay, this is likely to be a cloned amplicon.Create primary serial dilutions of your target We will assume that we are trying to determine the LoD of a qPCR reaction in terms of copies per reaction. Let’s demonstrate using the hypothetical example below. So how does one determine LoD? Luckily, there is a very straight forward approach: you create serial dilutions of your analyte and simply observe the rate of detection. If you subjected a sample with 15 copies per reaction to this assay, you might only detect the target 70% or 80% of the time. For instance, imagine if the LoD of your qPCR was 25 copies per reaction. You most certainly can, but it is just that much more improbable that you would. One of the most misunderstood facets of LoD is if you can detect an analyte at a level below your LoD. If you were calculating LoD for a quantitative polymerase chain reaction (qPCR), you might determine the lowest copy number of your target sequence that can be detected 95% of the time. See what I just did there? I added in probability! This is an important point – LoD is a probabilistic measurement. Quite simply, LoD is the lowest amount of a target or analyte that your assay can detect 95% of the time. samples that really contain the target of your assay). Sometimes, people will invoke the term sensitivity when they are talking about diagnostic sensitivity, but that refers to the ability of an assay to correctly identify true positive samples (i.e. Sensitivity in this context means analytical sensitivity, which is also known as limit of detection (LoD).
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Before we dig in, let’s get the terminology and definitions sorted. This is perhaps one of the most important performance characteristics you can determine for an assay, and in regulated work, such as in vitro diagnostic (IVD) development and clinical diagnostics, it is mandatory to do so. When developing an assay, whether it is for basic research or for use in diagnostics, you will often be asked about your assay’s sensitivity.