Multiplex real time PCR

A multiplex real time PCR reaction enables the amplification of more than one target in a single reaction, using different reporters.
Multiplex qPCR is a simple, efficient, and cost-effective solution for overcoming the challenges of limited samples and costly analysis.
The reliability of a multiplex PCR assay is affected by several factors including:
 
• Competition or inhibition between assays through interactions among the various primer pairs, probes, targets, amplicons, or any combination of these factors.
 
• The relative expression levels of targets (including endogenous controls), and the dynamic range of their expression.
 
Therefore, a reliable multiplex reaction should be well optimized.
General guidelines for optimization are available here.

 
• Multiplex your gene expression reaction – In gene expression multiplexing experiments, the goal is to minimize the difference between the Ct value in singleplex and multiplex reactions. Up to four targets can be multiplexed in a single reaction depending upon the probes that are selected. Reaction optimization should consider target abundance (read more). To convert your taqman assays for multiplexing look here and use the Multiplex Assay Conversion Tool.  

​See how Webb, Price, Somprasong et al. optimized a gene expression triplex reaction (Future Microbiol. (2018) 13(12), 1403–1418)
 
• Duplex your SNP reactions to detect two SNPs in one sample – It is recommended that duplex SNP reactions be performed using one TaqMan® Genotyping Assay (FAM™/VIC®) and one (ABY®/JUN®) SNP assay (read more).
To convert your taqman SNP assay for multiplexing use the Multiplex Assay Conversion Tool.
 
• Copy number variation (CNV) analysis – Run reference assays with copy number assays in duplex real-time PCR to detect and measure copy number variations in the human and mouse genomes. Use the Predesigned TaqMan Copy Number Assays for human, mouse and common vector marker and reporter. Use the designated CopyCaller® Software to determine CNV. 

 Pathogen detection in multiplex – A multiplex reaction can be used for qualitative detection of nucleic acid of viral or bacterial origin. Such detection method is found for example in the TaqPath™ COVID-19 Combo Kit for detection of nucleic acid from SARS‑CoV‑2.