How do the two forms of PCR Quantitation differ?

Honestly, this can be a very confusing area - especially since many of the controls used are shared between the categories, or made and characterised in the same way. So lets talk about each in turn remembering that their name pretty much describes the outcome.

One thing I'll say from the outset - to be absolutely sure your control is amplifying under the same conditions as your unknown, you need to either amplify them in the same tube, or else employ a reference that will indicate inter-tube variation caused by reagent loading errors. The former approach is subject to competition between the templates during the amplification process whilst the latter may not take into account variation in amplification efficiency Your standards should ideally amplify with the same efficiency. This is most likely to occurs when the templates are the same length, hybridise the same primers, have the same GC content, and any oligoprobes employed hybridise at the same position.

Relative quantitation
In this method we perform our PCR and compare the signal produced from our PCR product (amplicon), be it from gel electrophoresis, PCR-ELISA, or fluorogenic oligoprobe, to a control target. This target can be co-amplified or amplified in a separate tube, but in the same run. So this method describes the amount of template present, relative to a characterised control. "Characterised" takes on a fairly limited meaning in this instance. In this case it could simply contain the target of interest, or it could comprise a defined amount of target measured by mass, infectious particles or specific copies / genome equivalents. In these instances the test samples are positive if they produce an equal or higher signal and the degree of positivity or negativity can be obtained from the extent to which they fall above or below the control.

The relative control could also be a specimen collected from a subject (human, animal, plant or microbe) at a particular time during a disease, experimental or biological process or at an interesting time point in the lifespan of an organism.

Whatever the control, its role is to act as a threshold above or below which the results from test samples will fall. This tells us the state of the specimen and by inference, the host from which it came. Another assumption of course!

Absolute quantitation
This approach is more demanding because we have to try and control all the variables in order to return the exact number of target molecules present in our sample of interest. This is where the number of variables involved in a PCR experiment really become apparent.