Summary

TaqMan® Real Time RT-PCR represents the newest generation of expression analysis. PCR primers are used in conjunction with a specific TaqMan® probe, which is modified with a reporter fluorescent marker (FAM or VIC) at the 5' end and a light absorbing so-called quencher (i.e. TAMRA) at the 3' end. The quencher molecule absorbs the energy released from the neighbouring reporter, thus reducing the fluorescence emitted by the probe. During the extension phase of the PCR, the TaqMan® probe bound to the template is cleaved by the 5'-3' exonuclease activity of the Taq DNA polymerase. The resulting loss of spatial proximity between reporter and quencher leads to increased fluorescence (see figure).

This fluorescence can be measured in real time with the ABI PRISM 7500 real time PCR System (Applied Biosystems). The increase in reporter fluorescence is proportional to the amount of newly formed PCR product. The initial amount of DNA template can be calculated from the curve, which allows for a quantitative detection with a sensitivity of up to 5 molecules.

Typical applications for TaqMan® Real Time RT-PCR

• Validation of results from microarray expression analyses
• Expression profiling of specific mRNAs from different tissues
• mRNA expression pattern from small cell numbers
• mRNA expression pattern in primary cells from diseased organs

Our Service will include:

• development and setup of gene-specific TaqMan® Assays
• determination of optimal reference (housekeeping) genes for individual expression analyses
• measurements of established TaqMan® Assays in 20 tissues from mouse and human (Tissue Panel)

Additional Information

The PCR process and 5' nuclease process are covered by patents owned by members of the Roche Group. ABI PRISM is a registered trademark of PE Corporation. TaqMan® is a trademark of a member of the Roche Group. All other trademarks are properties of their respective owners.