Relative quantification of gene expression

This is also an easy optimization procedure, but one is looking for the best possible conditions of amplification (lowest Ct) with a similar PCR efficiency for both the target and the endogenous control(s) as reference

Forward primer (nM)

Reverse primer (nM)

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A standard protocol for optimizing primer concentrations.

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Figure 8.1B

Two sets of primers were designed to amplify genes grey and black. Primers for gene grey worked better at 300/300 nM (Ct 28), but those for gene black were better at 500/500 nM (Ct 25).

(usually several housekeeping genes). The procedure consists of finding the most efficient primer concentration for both genes (see above), followed by a comparison of efficiency between target and reference. The same optimization matrix can be set up to find appropriate primer concentration conditions (both for the target and the reference genes). Then, a serial dilution of the input cDNA is used to compare the efficiency of both PCR reactions in parallel. Ct values will be different between the target and the reference but one is looking for parallelism between the two standard curves. PCR efficiencies can be compared by relating the slope of the linear curve of Ct values plotted against the log-dilution. A perfect reaction has a slope of 3.3, which corresponds to an exact two-fold amplification at every cycle of the PCR between dilutions of a factor 10. An example of efficiency determination is shown in Figure 8.4.

Cycle number

Figure 8.2

An example of amplification plot.

Cycle number

Figure 8.2

An example of amplification plot.

Log dilution

Figure 8.3

An example of standard curve.

Log dilution

Figure 8.3

An example of standard curve.

Log dilution

Figure 8.4

Log dilution

Figure 8.4

Standard curves for different target sequences for comparative efficiency determination. Ideally they should be parallel (same slope) to be assayed together. The same serial dilution (from pure cDNA to 1/1,000,000) was used to analyze the efficiency of PCR reaction for genes square, diamond and circle against the housekeeping gene GAPDH (triangle). The efficiency for GAPDH was 3.20. It was equivalent to the efficiency of the PCR reaction for gene circle, 3.21. However, the PCR efficiency for gene square was good (3.19 for the first 4 serial dilutions); the PCR reaction reached its limit after the fourth dilution in a 40 cycles reaction. Finally, the efficiency of the PCR reaction for gene diamond was 3.78. This is too far apart for use in a relative quantification. A new set of primers should be designed for gene diamond.

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