Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 15)

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Determining the Real-Time PCR Performance Characteristics
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
1. A serial dilution of the cDNA, gDNA, or plasmid template is required to prepare the standard
curve. Ensure an adequate supply of template and an adequate volume are available to
evaluate all the assays used in the experiment.
2. Prepare the real-time PCR reactions using a fresh bottle of supermix, nuclease-free water,
and primer sets. Figure 6 is an example of a plate layout.
Fig. 6. Example of a plate layout with four seven-point
standard curves with NTCs in technical triplicates — one
for each gene of interest and the reference gene.
Reference
gene
Medium
expressor
Low
expressor
High
expressor
Fig. 5. Tenfold serial dilution covering six logs of dynamic range is prepared using a starting template
of your choice based on target expression levels.
1 1:10 1:100 1:1,000 1:10,000 1:100,000 1:1,000,000
Serial dilution of the template