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Duplexing in quantitative real-time PCR (qPCR) is the simultaneous detection of two target sequences in a single qPCR reaction.  Its key benefits come from minimizing the impact of pipetting errors and maximizing experimental efficiency.  Duplexing is only possible using TaqMan-based qPCR chemistry.  There is currently no method to duplex targets using SYBR Green qPCR chemistry.

Applied Biosystems TaqMan Gene Expression Assays are easily duplexed, since both predesigned and custom TaqMan Gene Expression Assays are available with either FAM or VIC fluorescent reporter dyes. The most common duplex reaction is combining the target of interest and endogenous control assays in the same well. An endogenous control gene or set of genes is typically used to control for experimental variability due to RNA input and shows expression levels that are relatively constant and moderately abundant across tissues, cell types, and treatment protocols.

Why duplex?

Precious samples—when cDNA is limited, duplexing allows more targets to be analyzed since two targets are measured using a single input of cDNA

Cost—run half the number of reactions compared to singleplex qPCR, thereby using half as much master mix and fewer plastic consumables

Precision—data quality is improved because pipetting variability is minimized when the target of interest and endogenous control are measured within the same well

Normalization—use an endogenous control assay in the same well as your target of interest to normalize the expression results and correct for any potential biases caused by the amount of RNA input, reverse transcription efficiency, or sample quality

Learn more about choosing endogenous controls for your experiment ›

Find predesigned TaqMan Gene Expression Assay ›


Support for duplexing TaqMan Gene Expression Assays

Assay formats

Our predesigned TaqMan Gene Expression Assays come in three different formats: FAM, VIC, and VIC_PL.

Assays are labeled with either a FAM or VIC fluorescent dye.  For targets that are highly expressed (e.g., 18S rRNA, the common endogenous control), we recommend choosing our VIC-primer-limited (VIC_PL) format, which has a decreased amount of primers to limit the housekeeping gene from consuming the qPCR reagents before the target of interest can amplify.

TaqMan Gene Expression Assay, FAM
TaqMan Gene Expression Assay, VIC
TaqMan Gene Expression Assay, VIC primer-limited

Resources 

Application Note: Duplexing in quantitative real-time PCR with TaqMan Gene Expression Assays

Contact Technical Support to check for duplexing compatibility of TaqMan Gene Expression Assays.

TaqMan Fast Advanced Master Mix has been optimized for duplex qPCR.

For information related to higher level multiplex qPCR (3- and 4-plex reactions), please consult our Multiplexing qPCR solutions webpage.

Related application

Gene Expression Analysis

Data analysis tools

Connect cloud-based platform—free to sign up
Desktop software—downloads can be found in the Support Center for your specific Applied Biosystems qPCR instrument
ExpressionSuite Software—free to download