Standardisation of data from real-time quantitative PCR methods - evaluation of outliers and comparison of calibration curves.
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.


CONCLUSIONS
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.
Burns
Malcolm J
MJ
Bio-Molecular Innovation, LGC Limited, Queens Road, Teddington, Middlesex, TW11 0LY, UK. malcolm.burns@lgc.co.uk
Nixon
Gavin J
GJ
Foy
Carole A
CA
Harris
Neil
N
D001709:Biotechnology / Q000379:methods*; D002138:Calibration; D019411:Clinical Laboratory Techniques; D017931:DNA Primers; D003627:Data Interpretation, Statistical; D005069:Evaluation Studies as Topic; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006801:Humans; D015233:Models, Statistical; D008962:Models, Theoretical; D012015:Reference Standards; D012044:Regression Analysis; D015203:Reproducibility of Results; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012984:Software
D017931:DNA Primers; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)