( employed. Small differences in measured...)Amplification Efficiency of TaqMan Gene Expression Assays

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

Use a DNA template with high enough target template concentration to create a 56 log dilution series that will generate C_T 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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