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Amplification Efficiency of TaqMan Gene Expression Assays

employed. Small differences in measured CT values can translate into significant changes in calculated efficiency, especially when few points over a narrow dilution range are used to generate the CT vs. log concentration plot, and thus calculate a CT slope.

When using custom designed TaqMan probe and primer sets that are not a part of the TaqMan Gene Expression Assays product line, we recommend:

To obtain reliable efficiency values, use a 56 log (105106 fold) dilution range of template DNA

Use at least 5 data points in this dilution range to generate the CT versus log concentration plot

Use a DNA template with high enough target template concentration to create a 56 log dilution series that will generate CT values of less than 36 on an Applied Biosystems real-time PCR instrument.

We suggest you use:

PCR product (be very careful to avoid contamination of the laboratory space with amplified DNA);

A cDNA clone at a high template concentration; or

Genomic DNA (if your TaqMan probes and primers are designed within an exon)

References

1 Livak and Schmittgen, (2001) Methods 25:402408

2 Liu & Saint, (2002) Analytical Biochemistry, 302:5259

3 Relative Quantitation of Gene Expression: ABI PRISM 7700 Sequence Detection System Livak (1997), Applied Biosystems User Bulletin #2 (part number 4303859)

Additional Recommended Reading

Essentials of Real-Time PCR Applied Biosystems Application Note (part number 105622)


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