The figure below shows the results of a “real-time PCR” experiment. The primers

Question

The figure below shows the results of a “real-time PCR” experiment. The primers employed for this experiment target a so-called “housekeeping” gene. Based on this figure, answer the following questions.

Label the y-axis.

To what does the word “Cycles” refer?

What is the blue horizontal line depicting and how do you use it to understand these results?

Given the information you have, you cannot tell whether the experiment is measuring the gene (i.e., DNA) or the messenger RNA. What would you need to know about the way the experiment was performed to distinguish between the two?

Explanation / Answer

y-axis represents Relative floresence.

The Cq value or cycle quantification value is the PCR cycle number at which your sample's reaction curve intersects the threshold line.

Horizontal line represents ground phase,only background fluorescence is detected. The cycle at which the amplification fluorescence exceeds a chosen threshold above the background fluorescence is called the Cycle threshold or Ct value and this marks the early exponential phase. It is important to quantitate the initial DNA copy number at this time as it will be the most accurate

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