Duplexing in quantitative real-time PCR (qPCR) involves detecting two target sequences simultaneously within a single qPCR reaction. This technique offers significant benefits, such as reducing pipetting errors and increasing experimental efficiency. Duplexing with TaqMan-based qPCR chemistry allows more specificity compared to SYBR Green qPCR chemistry.
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 experimental variability due to RNA input and shows expression levels that are relatively constant and moderately abundant across tissues, cell types, and treatment protocols.
When to use duplex qPCR assays
- Precious samples—When cDNA is limited, duplexing allows more targets to be analyzed since two targets are measured using a single sample of cDNA
- To reduce cost—Run half the number of reactions compared to singleplex qPCR, thereby using half as much master mix and fewer plastic consumables
- To increase precision—Data quality can be improved because pipetting variability is minimized when the target of interest and endogenous control are measured within the same well
- To normalize expression data—Use an endogenous control assay in the same well as your target of interest to normalize the expression results and correct for potential biases caused by the amount of RNA input, reverse transcription efficiency, or sample quality
Learn more:Choosing endogenous controls
Formats for duplexing TaqMan gene expression assays
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 quantity of primers to limit the housekeeping gene from consuming the qPCR reagents before the target of interest can be amplified.
Resources for duplexing TaqMan gene expression assays
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.
For information related to higher level multiplex qPCR (3- and 4-plex reactions), please consult Multiplexing qPCR Solutions.
Data analysis tools for duplexing TaqMan gene expression assays
Learn more: Applied Biosystem qPCR analysis modules
Learn more:Digital solutions for scientific ecosystems
Desktop software—Downloads can be found in the Support Center for your specific Applied Biosystems qPCR instrument.
Related products and applications for duplexing TaqMan gene expression assays
Learn more: TaqMan Endogenous Controls
Learn more:MicroRNA and non-coding RNA applications for qPCR