What is PCR Quantitation?

PCR has traditionally been used as a tool for visualising very small amounts of a specific piece of target RNA or DNA. This is the method of qualitative PCR used to confirm the presence of that piece of nucleic acid in our test sample. PCR can also be used to tell us the amount of a particular piece of RNA or DNA originally present in our sample. We say we can quantify how much of the particular NA is in the sample, and in doing so we quantitate that NA, or perform NA quantitation. Quantitative PCR (qPCR) is a powerful tool and many practical uses have been identified in recent years. These can be broadly broken into two main categories and several sub-categories:

Human genome derived applications:

• Gene expression studies;
• Mutation analysis;
• Cancer marker monitoring;
• Drug response monitoring;
• Residual cell/DNA monitoring;
• Gene therapy construct/transgene analysis
  

Non-human applications:

• Microbial load studies;
  
• Monitoring response to antimicrobial therapies;
• Gene therapy vector analysis

NA quantitation can be performed relative to a single, well-characterised standard or by determining the absolute number of target molecules. The latter is more technically demanding whilst the former is more common and generally provides all the information needed.

But its not that easy! As with any laboratory tool, qPCR is only as good as the controls used to define its results. I'll talk more about these controls in the following pages and hopefully provide some examples to illustrate their use. Its well worth remembering that the majority of publications quoting the use of "quantitative PCR" are more likely to be using semi-quantitative PCR because one or more essential variables have not been controlled accurately or described completely. Highly accurate qPCR is very difficult to achieve, whilst precise and reproducible qPCR is relatively easy. So before embarking upon this path - make sure you are confident that your experimental design is top notch, otherwise your results won't mean what you expect them too!

Despite the relative maturity of conventional PCR techniques, it is increasingly difficult to find answers to simple questions so this section of VDU will aim to provide as much basic information as possible. We'll also go into conventional competitive qPCR techniques from which we can formulate a list highlighting the to be addressed by the next evolution of PCR, real-time PCR (rtPCR).

One last point - real-time PCR DOES NOT MEAN quantitative PCR! There are a lot of controls and experimental considerations required for conventional or rtPCR to accurately quantify NA. Please don't assume that the former means the latter.

Controls

Relative vs Absolute