ABSTRACT
The original limited options of detection chemistry (SYBR Green I-binding dye or 5′-nuclease assay), enzyme (Taq polymerase), and instrument (Lightcycler® or ABI 7700) have been replaced with extensive alternatives of methodology, methods, instruments, and reagents, giving rise to numerous protocol choices with the potential to generate technique-dependent conicting data.5-8 Any underlying tendency toward inconsistency is aggravated by the signicant probability of variability associated with the numerous steps that make up a qPCR assay9 (Figure 15.1). Preanalysis variability derives from issues such as poorly dened sample selection or handling, patchy nucleic acid quality, inconsistent use of controls, poor assay design, as well as nonexistent optimization and validation. Inconsistencies with post-qPCR data processing are caused by a lack of quality control of the qPCR data, especially unsuitable methods of normalization, misguided data-analysis procedures, and challenges associated with applying the correct statistical methods.10
