How to Optimize Multiplex qPCR Experiments -- Ask TaqMan® Ep. 18 скачать в хорошем качестве

How to Optimize Multiplex qPCR Experiments -- Ask TaqMan® Ep. 18

Submit your real-time PCR questions at https://www.thermofisher.com/us/en/ho... In this video, Sr. Field Applications Specialist Doug Rains explores the basic idea behind multiplexing gene expression experiments. Learn about central primer-limitation strategy for optimizing such an experiment and the necessary steps for validating the accuracy of a multiplex scenario. TaqMan is a robust, highly specific, and -- might I add -- rather flexible real-time PCR chemistry. Users have the option to label their TaqMan probes with different dyes, permitting them to multiplex more than one target in a single well. In a related Ask TaqMan video, I discuss the advantages of multiplexing. In this video, I'll discuss a big danger of this technique, as well as answer a related inquiry from Rushita at Institute of Cardiovascular Sciences in Canada, namely: "What is the best and cost-effective way to standardize a multiplex experiment?" Let's quickly review some basics. In a singleplex gene expression experiment, I always amplify my target and endogenous control assays in separate wells. But say I want to pipette these into a single well. To do so, I need to label my two assay probes with two different dyes that my instrument can distinguish. Let's go with FAM and VIC. At this point, I simply combine my two probes into a single well, amplify, and get comparable data to my sinlgeplex experiment. Right? Maybe. Here's the concern... When 2 assays share a well, they compete for the same pool of common reagents, including enzyme and dNTPs. If one gene, typically the normalizer, comes up much earlier in the reaction, it may hit its linear and plateau phases before the second gene even appears in our amp plot. The target gene will thus be starved of common reagents, such as dNTPs, and likely experience poor amplification. Fortunately, there's an adjustment we can make that often solves this problem: namely, primer-limiting the normalizer gene. If we significantly reduce the normalizer assay's primer concentration, it will hit its plateau much earlier -- not because it's used up the dNTPs, but rather, its own primer. In theory -- and often in practice -- enough common reagent will remain in the well for the target gene assay to amplify properly. Thermo Fisher sells an array of VIC-labeled, primer-limited endogenous control assays for duplex gene expression experiments. I suggest starting with one of these for duplex gene expression. That said, you should still run some tests to make certain that combining assays doesn't affect final results. My suggestion? Take several of your samples -- maybe 5 or 6 untreated, plus 5 or 6 treated -- and amplify them all with both of your assays in singleplex and in duplex. Next, calculate fold change data. If sample-to-sample results agree between your two groups, it's likely that multiplexing isn't having a negative effect on your results. But if they don't agree, you're probably better off not taking the risk. One last technical point: Most real-time instruments are equipped with multiple filter sets, suggesting one can combine numerous assays in a single well. But be very careful here: the more targets one adds to a single reaction, the more complex the interactions among them, and the greater the danger that final data will be skewed. Remember: always validate any multiplex by comparing its results across several samples, and even over a range of starting RNA concentrations -- just to be sure your multiplex data are trustworthy. If you'd like to learn more about real-time multiplexing, please go to ThermoFisher.com and search for the application note entitled, "Factors Influencing Multiplex Real-Time PCR."