Determining the pcr efficiency, Determining the pcr efficiency 11, Pcr efficiency – Bio-Rad SsoAdvanced™ Universal Probes Supermix User Manual

Page 17: Dynamic range, Correlation coefficient, Sensitivity, Specificity

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SsoAdvanced

™

Universal Probes Supermix Instruction Manual

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Determining the PCR Efficiency

Determining the PCR efficiencies of your reference gene and target gene(s) is critical before
starting any real-time PCR experiment. Knowing the PCR efficiency determines the appropriate
relative gene expression math model. Not knowing may affect and invalidate the results. To
determine the PCR efficiency among other key characteristics, prepare standard curves to
evaluate the following:

■

PCR efficiency

■

Dynamic range

■

Correlation coefficient

■

Sensitivity

■

Specificity

Fig. 6. The blue standard curve represents the target gene and the green standard curve
represents the reference gene. A, both assays demonstrate equivalent performance in linearity

and dynamic range covering 1 µg to 1 pg. Thus, any RNA input going forward within this range will
be acceptable; B, both assays are either saturated at the 1 µg data point or the reverse transcription

reaction is inhibited due to carryover inhibitors from the RNA sample. Consider using less RNA (≤100 ng)
or re-purifying the RNA; C, the reference assay has a broader dynamic range than the target assay,

therefore, the dynamic range is limited. Consider reevaluating the target assay design, using less RNA
(≤100 ng), or re-purifying the RNA; D, the target assay exhibits a high standard deviation at the lowest

concentration (1 pg) and should not be considered part of the dynamic range. This is due to a lack of
sensitivity or reproducibility, and may be alleviated by using a carrier in the RNA sample such as
glycogen or non-target gDNA carrier; E, after considering all the data, the concentration points that

define the dynamic range from rejecting the variant 1 pg data and the saturated/inhibited 1 µg data point
results in an effective dynamic range (RNA loading) is 1–100 ng.

Initial RNA

1 pg

10 pg

100 pg

1 ng

1 µg

10 ng

100 ng

1 pg

10 pg

100 pg

1 ng

1 µg