ABSTRACT
The specificity, wide dynamic range and ease-of-use of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has made it the method of choice for quantitating RNA levels. However, as with any scientific measurement, qRT-PCR suffers from problems caused by error (Huggett et al., 2005). Consequently, data normalization is an indispensable component of the qRT-PCR analysis process, and is essential for accurate comparison of qRT-PCR measurements between different samples. Normalization controls for variation in the total mass of RNA analyzed and, ideally, corrects for any biological and technical variability. Both of these pose serious problems for gene expression analyses and manifest themselves as noise that can either mask or overstate true expression levels and patterns. It is vexing that whilst the requirement to incorporate some normalization method to control for error is obvious, in practice it remains one of the most discussed, but unsolved problems of qRT-PCR analysis (Huggett et al., 2005). This chapter will outline normalization, address the associated problems when using real-time PCR and discuss data interpretation once normalization has been performed.
