qPCR analysis

The qPCR results consist of a melting curve and an amplification plot.

• The melting curve shows at what temperature the DNA strands melt/separate. This is very useful to evaluate if you are measuring the amplification of your transcript alone or if you have contaminants.

• The amplification plot depicts the change in fluorescence as log (ΔRn) in each PCR cycle.

The cycle at which the fluorescence crosses a specified threshold is called the Ct value (also called Cq for quantification cycle). A low Ct indicates a high amount of initial transcript, as it took only a few cycles to reach the fluorescence threshold, and therefore a high expression of the investigated gene. A high Ct value will indicate small amounts of initial transcript, as it required a high amount of amplification cycles to reach the fluorescence threshold, and therefore low expression of the gene.

At first, one should check if the negative controls produced a signal. If there is a signal for these water controls, the experiment is contaminated with foreign DNA and the results are not valid.

There are different methods to calculate the relative amounts of each DNA from the measured fluorescence. The most common method is the delta delta Ct method.