The fluorescence-based quantitative real-time PCR (qPCR) (15) (1-3), with its capacity to detect and measure minute amounts of nucleic acids in a wide range of samples from numerous sources, is the enabling technology par excellence of molecular diagnostics, life sciences, agriculture, and medicine (4, 5). Its conceptual and practical simplicity, together with its combination of speed, sensitivity, and specificity in a homogeneous assay, have made it the touchstone for nucleic acid quantification. In addition to its use as a research tool, many diagnostic applications have been developed, including microbial quantification, gene dosage determination, identification of transgenes in genetically modified foods, risk assessment of cancer recurrence, and applications for forensic use (6-11). This popularity is reflected in the prodigious number of publications reporting qPCR data, which invariably use diverse reagents, protocols, analysis methods, and reporting formats. This remarkable lack of consensus on how best to perform qPCR experiments has the adverse consequence of perpetuating a string of serious shortcomings that encumber its status as an independent yardstick (12). Technical deficiencies that affect assay performance include the following: (a) inadequate sample storage, preparation, and nucleic acid quality, yielding highly variable results; (b) poor choice of reverse-transcription primers and primers and probes for the PCR, leading to inefficient and less-than-robust assay performance; and (c) inappropriate data and statistical analyses, generating results that can be highly misleading. Consequently, there is the real danger of the scientific literature being corrupted with a multitude of publications reporting inadequate and conflicting results (13). The publication (14) and retraction (15) of a Science "Breakthrough of the Year 2005" report provides a disquieting warning. The problem is exacerbated by the lack of information that characterizes most reports of studies that have used this technology, with many publications not providing sufficient experimental detail to permit the reader to critically evaluate the quality of the results presented or to repeat the experiments. Specifically, information about sample acquisition and handling, RNA quality and integrity, reverse-transcription details, PCR efficiencies, and analysis parameters are frequently omitted, whereas sample normalization is habitually carried out against single reference genes without adequate justification.
