Editing QPCR mix (section)

=== Using the 2<sup>-ΔΔC<sub>T</sub></sup> Method ===

This method uses a second set of primers which anneal to a greater population of DNA as an reference. This eliminates the vagueness of the total DNA method and the error introduced by differential DNA extraction.

*The amount of the target DNA relative to the reference DNA is 2<sup>-ΔC<sub>T</sub></sup> where ΔC<sub>T</sub> = ΔC<sub>T, target</sub> - ΔC<sub>T, reference</sub>.
*Typical output feels like "My population is 25% percent of the total bacterial population".
*You actually still have to make a standard curve (and sometimes more than one) for two reasons:

:1. To be sure that your two primer pairs are amplifying at the same efficiency.
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*The ΔC<sub>T</sub> values should be approximately the same for all dilutions.
::*This standard curve can be done with DNA from any sample and need not necessarilly be from a pure culture.
:2. To calibrate the relationship between the two amplicons.
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*If your comparing a one copy gene (like an enzyme) to a many copy gene (like rRNA genes) then a numerical relationship needs to be established between them.
::*If your amplicons are different lengths then this will also be taken care of in this calibration.
::*Sometimes it helps to use a DNA from pure cultures of different genera to determine this relationship if that's important to you.