Welcome to Ask TaqMan. Today’s real-time PCR question: What’s a threshold and where do I place it? Excellent question. The threshold is a horizontal line in our amplification plot that can be moved up or down on the y-axis. It’s purpose, as we’ll see in a minute, it tells the software where to take data. Now, not all places on the y-Axis are equal. Some regions we want to avoid – specifically we don’t want to be too low, otherwise we get down into the noise. Conversely, if we go too high, we’re in the linear or plateau phase of amplification where data are less predictable. A happy spot? Someplace where all of our curves are straight and parallel to one another. What we really want is to put the threshold wherever the precision of our replicates is highest. That’s generally somewhere toward the middle of the geometric phase or maybe slightly higher. In any case with a really robust assay hitting a good spot is quite easy. The default on all Applied Biosystems real-time PCR software is auto threshold meaning the software sets thresholds for us the second we click analyze. Notice that it sets a different threshold for each assay separately, which is good since not all assays have the same sweet spot. If I want though, I can switch any one or all of my thresholds to manual mode. Then move the line up or down with my mouse. Once the threshold is set and we click analyze all the samples get their respective Ct values. The attentive viewer might be tempted to ask well if the threshold can be moved up or down, doesn’t that change Cts? Why yes it does. But here’s the thing: as long as we keep the threshold firmly within the geometric phase, the relative or delta Ct between any two samples stays constant. This fact allows us to do things like calculate fold changes in gene expression from sample to sample, and to get quantity information from a standard curve That’s all there is to threshold. If you have any real-time PCR questions, just ask TaqMan. Literally! You can send your question via Twitter by using the hashtag Ask TaqMan, post to our Facebook timeline, or just visit lifetechnologies.com/asktaqman And hey, don’t forget to check out the rest of our Ask TaqMan videos available on YouTube.