Improved Indicators for Assessing the Reliability of Detection and Quantification by Kinetic PCR
2003; American Association for Clinical Chemistry; Volume: 49; Issue: 6 Linguagem: Inglês
10.1373/49.6.973
ISSN1530-8561
Autores Tópico(s)RNA Research and Splicing
ResumoBecause of their low detection limits, PCR and the adapted kinetic (real-time quantitative) PCR have been used extensively for the detection and quantification of nucleic acids (1)(2)(3). However, at the detection limits of an assay, mathematical models (4)(5)(6) and experimental evidence (7)(8)(9)(10)(11)(12) have shown that nucleic acids are detected inconsistently and quantified imprecisely. With this potential unreliability, it is essential that indicators be used to identify the concentration at which analysis is occurring to ensure the accuracy of results. Conventional PCR indicators, however, currently provide only an indirect assessment of the concentration at which analysis occurs. The total nucleic acid content of a sample, commonly used to define the adequacy of sample loading, does not precisely reflect specific gene concentrations. Reference (control or housekeeping) genes, qualitative detection of which is often used to validate assay and sample integrity, are often present at concentrations different from those of the target genes of interest (13). In kinetic PCR, the inclusion of fluorescent probes allows the monitoring of reaction kinetics, which enables the measurement of a crossing point (CP), or cycle threshold, at a partial cycle number at which fluorescence becomes detectable above background signal (14). CP values are linearly proportional to gene copy number in an inverse logarithmic relationship (14)(15), and this correlation is central to algorithms for determining gene quantity (16)(17). In this study, we hypothesized that CP values might be more accurate indicators of the reliability of an analysis than conventional indicators because of their direct relationship with gene concentration. The results of this study provide experimental evidence to support this hypothesis; we also describe models for use of CP values as indicators for assessing the reliability of …
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