An Inter-Platform Repeatability Study Investigating Real-Time Amplification of Plasmid DNA

Published

May 25, 2005

Author(s)

C Donald, F Qureshi, M J. Burns, Marcia J. Holden, J L. Blasic, A J. Woolford

Abstract

Background: Currently, there are many different real-time amplification platforms. However, the results of DNA measurements from experiments performed on different platforms are not necessarily comparable and results may not be repeatable within instruments.Methods: On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms the LightCycler , ABI PRISM 7700 and Rotor Gene 3000 . Real-time PCR amplification using a fluorogenic 5 exonuclease assay was performed in triplicate on DNA plasmid dilutions of 108-102 copies and on negative controls to give a total of 24 reactions per PCR experiment. Statistical analysis of the raw data was undertaken to assess intra-platform repeatability.Results: Statistical Univariate tests indicated that the most repeatable platform in this trial was the ABI PRISM 7700 when coupled with the FastStart PCR reaction mix. It was also found that there was no obvious relationship between plasmid copy number and repeatability. An ANOVA analysis identified the factors which significantly affected the results: plasmid copy number, platform, PCR reaction mix and day (on which the experiment was performed), in descending order of magnitude.Conclusions: In order to deliver useful, informative genetic tests, standardisation of real-time PCR detection platforms to provide repeatable, reliable results is warranted alongside a better understanding of inter-assay and intra-assay repeatability.

Citation

Bmc Biotechnology

Volume

5

Pub Type

Journals

Keywords

equipment comparability, quantitative PCR, repeatability

Citation

Donald, C. , Qureshi, F. , Burns, M. , Holden, M. , Blasic, J. and Woolford, A. (2005), An Inter-Platform Repeatability Study Investigating Real-Time Amplification of Plasmid DNA, Bmc Biotechnology (Accessed April 20, 2024)