Real-Time PCR Thresholds and Where to Place Them -- Ask TaqMan®: Ep. 6 скачать в хорошем качестве

Real-Time PCR Thresholds and Where to Place Them -- Ask TaqMan®: Ep. 6

Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding of thresholds in Real-Time PCR. The threshold is a horizontal line in our amplification plot that can be moved up or down on the Y-axis. Its purpose? As we'll see in a minute, it tells the software where to take data. Of course, not all places on the Y-axis are equal. Some places 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? Some place 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 bad spot is quite difficult. 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. I could go 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. Now, the attentive viewer might be tempted to ask: if the threshold can be moved up or down, doesn't that change the Cts? The answer is, "Yes." 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 expression from sample to sample, and to get quantity information from a standard curve.