Data analysis

The goal of real time PCR is typically to quantify the relative ratio between two templates. These templates might be

  • two different mRNA transcripts (to look at relative gene expression)
  • the quantify of one mRNA transcript under two different treatments (two different life stage)
  • two different DNA templates (to compare the presence of two different species in a population).

    • For these comparisons, the two templates are prepared in exactly the same way (e.g. reverse transcribed using the same amount of total RNA). During real time PCR, the amount that they amplify can be compared and used to quantitate one to the other.

    The amount of PCR fluorescence, R, for a given sample is related to the several factors: the amount of template before PCR begins (T), the number of PCR cycles (n), and the efficiency with which PCR occurs each cycle (E). The fluorescence as a function of PCR cycles is then given by:

    Rn = k T (1+E)n
    where k is just a constant.

    At threshold, you set a threshold level and determine the critical cycle number (Ct) at which each sample crosses this threshold. At this point, both samples have the same amount of fluorescence equal to the threshold level (Rth). Then the following equation holds:

    Rth = k T1 (1+E1)Ct1 =k T2 (1+E2)Ct2

    To determine the ratio between the two templates, this equation is rearranged:
    T2 / T1 = (1+E1)Ct1 / (1+E2)Ct2

    The real time machine gives you Ct1 and Ct2 (the critical cycle numbers for templates 1 and 2). However, to accurately determine this ratio, you also need the efficiencies for the primer / probe sets corresponding to each template.

    If you know that your efficiencies are the same (see User bulletin#2 from ABI for ways to check this), this further simplifies to

    T2 / T1 = (1+E)Ct1-Ct2
    This delta Ct method is often used to get ratios from differences in cycle number.