Roche Life Science | Probes and The Universal ProbeLibrary

Multiplex Assays

Multiplex Assays with Universal ProbeLibrary Reference Gene Assays

Use the Universal ProbeLibrary Reference Gene Assays together with the Universal ProbeLibrary to easily quantify expression levels of a human, mouse, or rat gene of interest in relation to an endogenous reference gene in a dual-color assay. Three reference gene assays are available for human (G6PDH, GAPD and ACTB- gene), 2 for mouse (ACTB-, and GAPD gene each).

Each Universal ProbeLibrary Reference Gene Assay provides a specifically designed 12-mer UPL reference gene probe and the corresponding reference gene-specific primer pair in a separate tube. The probe is labeled with LightCycler®Yellow 555 at the 5´ end and with a dark quencher dye near the 3´ end, to enable dual-color assays together with the standard UPL probes, which are labeled with FAM. The UPL reference gene probes can be detected using real-time PCR instruments with excitation filters of 470 nm to 530 nm and emission filters of 550 nm to 610 nm. In multiplex assays, a standard FAM or SYBR® Green I filter is used as the first channel to detect FAM-labeled UPL probes for the gene of interest, and a longer-wavelength emission filter (usually the next possible emission filter moving to longer wavelengths, e.g., 560 nm or 568 nm) is used as the second channel to detect UPL reference gene probes.

Multiplex PCR assays for a target gene and a UPL Reference Gene are designed by the ProbeFinder software at the Assay Design Center with a high success rate of over 90%. In case no matching assays are found for the reference gene of choice (out of the listed assays), ProbeFinder SW suggests dual-color assays with one or more other reference genes out of the list.

Designing multiplex assays is as easy as designing monoplex assays: when you select the multiplex option online, ProbeFinder will design multiple UPL assays for your gene of interest. At the same time, each assay is subjected to in silico PCR testing to verify that it can be multiplexed with one (or more) of the UPL Reference Gene Assays for the relevant organism.


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