Malcolm Burns

Abstract Background Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated. Results At very low target concentrations of 0.5–10 genome equivalents (g.e.) eliminating any replicates within each DNA standard concentration with no measurable signal (non-detects) compromised calibration. Improved calibration could be achieved by eliminating all calibration replicates for any calibration standard concentration with non-detects ('elimination by sample'). Test samples also showed positive bias if non-detects were removed prior to averaging; less biased results were obtained by converting to concentration, including non-detects as zero concentration, and averaging all values. Tube plastic proved to have a strongly significant effect on DNA quantitation at low levels ( p = 1.8 × 10 -4 ). At low concentrations (under 10 g.e.), results for assays prepared in standard plastic were reduced by about 50% compared to the low-retention plastic. Preparation solution (carrier DNA or stabiliser) was not found to have a significant effect in this study. Detection probabilities were calculated using logistic regression. Logistic regression over large concentration ranges proved sensitive to non-detected replicate reactions due to amplification failure at high concentrations; the effect could be reduced by regression against log (concentration) or, better, by eliminating invalid responses. Conclusion Use of low-retention plastic tubes is advised for quantification of DNA solutions at levels below 100 g.e. For low-level calibration using linear least squares, it is better to eliminate the entire replicate group for any standard that shows non-detects reasonably attributable to sampling effects than to either eliminate non-detects or to assign arbitrary high Ct values. In calculating concentrations for low-level test samples with non-detects, concentrations should be calculated for each replicate, zero concentration assigned to non-detects, and all resulting concentration values averaged. Logistic regression is a useful method of estimating detection probability at low DNA concentrations.

BACKGROUND: As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. RESULTS: Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. CONCLUSION: The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will help contribute towards the development of a set of best practice guidelines regarding standardising handling and interpretation of data arising from RT-QPCR experiments.

BACKGROUND: In 2009, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines established standards for the design, execution, and reporting of quantitative PCR (qPCR) in research. The expansion of qPCR into numerous new domains has driven the development of new reagents, methods, consumables, and instruments, requiring revisions to best practices that are tailored to the evolving complexities of contemporary qPCR applications. CONTENT: Transparent, clear, and comprehensive description and reporting of all experimental details are necessary to ensure the repeatability and reproducibility of qPCR results. These revised MIQE guidelines reflect recent advances in qPCR technology, offering clear recommendations for sample handling, assay design, and validation, along with guidance on qPCR data analysis. Instrument manufacturers are encouraged to enable the export of raw data to facilitate thorough analyses and re-evaluation by manuscript reviewers and interested researchers. The guidelines emphasize that quantification cycle (Cq) values should be converted into efficiency-corrected target quantities and reported with prediction intervals, along with detection limits and dynamic ranges for each target, based on the chosen quantification method. Additionally, best practices for normalization and quality control are outlined and reporting requirements have been clarified and streamlined. The aim is to encourage researchers to provide all necessary information without undue burden, thereby promoting more rigorous and reproducible qPCR research. SUMMARY: Building on the collaborative efforts of an international team of researchers, we present updates, simplifications, and new recommendations to the original MIQE guidelines, designed to maintain their relevance and applicability in the context of emerging technologies and evolving qPCR applications.