b'One strategy that ensures high-quality PCR data is thatDigital PCR methods and toolsinvestigators follow standardized practices for performingDigital PCR (dPCR) is a method that quantifies sequences and reporting qPCR results. The MIQE (Minimum Informationpresent in a sample by counting the number of copies of the for Publication of Quantitative Real-Time PCR Experiments)target sequence. The basis of dPCR is that a nucleic acid recommendations are set of guidelines for qPCR experimentalsample is physically compartmentalized into thousands of designs and data reporting practices, as well as standardsparallel PCR reactions, such that each reaction well contains for sharing experimental information with colleagues [16]. Theone target molecule on average (Figure 6). In this scenario, standards are designed to ensure that published qPCR datasome reactions may not contain the target molecule at all, while is meaningful, accurate, and provides researchers with theothers will contain one or more copies. The collection of these information necessary to faithfully reproduce results. Thermocompartmentalized reactions is subjected to endpoint PCR, Fisher supports these guidelines and provides all informationand the number of wells with a positive signal and no signal necessary to ensure MIQE compliance when publishingare tallied. The number of target copies is calculated from the the results of experiments with TaqMan assays. Informationfraction of negative reactions, based on the assumption that the requested under the qPCR target, qPCR oligonucleotide, andsegregation follows a Poisson distribution (thus accounting for qPCR protocol sections of the guidelines are readily available inthe possibility that multiple target molecules occupy the same the reagent protocol or from our website.reaction). The number of individual reactions influences the The TaqMan Assay portfolio is made up of over 20 millionsensitivity of the assaythe more reactions there are, the lower predesigned assays. These assays can be ordered as individualthe limit of detection and the higher the accuracy. assays in tubes. Alternatively, TaqMan arrays contain TaqMandPCR is often used to detect rare mutant alleles in cancer Assays dried down in three array formats: 384-well TaqMan arraysamples and is used to analyze circulating tumor DNA (ctDNA) microfluidic cards (TACs), 96-well and 384-well TaqMan arrayin liquid biopsy research. Because the entire sample is plates, and Applied Biosystems OpenArray formats.compartmentalized into individual wells, the detection of rare To help with choosing the right TaqMan Assays for analleles is not masked by an overabundance of normal alleles. experiment, we developed an assay search wizard (link).The sensitivity is dependent on the amount of DNA; to achieve a Querying the gene name, keyword, pathway, or disease cansensitivity of 0.1% (1 in 1,000 copies), 1,000 copies are needed. return a list of all the assays that meet the search criteria. TheFor diploid genomic DNA (gDNA), at least 6 ng of input gDNA is results page highlights the best coverage assay for each gene,required for this sensitivity. Thus, the amount of recoverable DNA publications that used the assay highlighted, and provides anlimits the sensitivity of the assay.opportunity to select and design a combination of assays for anThe Applied Biosystems QuantStudio Absolute Q Digital array format. Furthermore, predesigned arrays are available thatPCR System consolidates all workflow steps into a single plate, have been prespotted with gene expression assays targetingtransforming a multi-step, multi-instrument workflow into a common pathways, diseases, and gene families, including severalone-step qPCR-like workflow. Absolute Q Liquid Biopsy dPCR that are important in cancer research. Assays on the QuantStudio Absolute Q dPCR system detect and Preparation Digitization Amplication Quantitation25k20kFluorescence15k10k5k00 5k 10k 15k 20kMicro chamberFigure 6. Basics of a digital PCR assay. dPCR reactions on the QuantStudio Absolute Q dPCR system make use of Absolute Q dPCR assays (TaqMan chemistry) to detect a target sequence. In dPCR assays, the sample is diluted and loaded into a matrix containing thousands of individual reaction chambers. If the dilution is correct, some of the chambers will have a target and some will not. Subjecting the chambers to PCR will cause a positive signal in the chambers where there is a target, but no signal where there is no target. The wells that are positive and negative are counted, and the starting concentration can be calculated based on dilution and other factors. Because the sample is loaded such that there is one molecule per microchamber, an overabundance of one sequence will not necessarily overwhelm detection in wells that have a rare sequence.Contents 19'