Journal Information
Journal ID (publisher-id): BM
Journal ID (nlm-ta): Biochem Med
Title: Biochemia Medica
Abbreviated Title: Biochem. Med.
ISSN (print): 1330-0962
ISSN (electronic): 1846-7482
Publisher: Croatian Society of Medical Biochemistry and Laboratory Medicine
Article Information
Copyright: 2016, Croatian Society of Medical Biochemistry
Date received: 20 April 2015
Date accepted: 19 November 2015
Publication date (print and electronic): 15 February 2016
Volume: 26
Issue: 1
Pages: 103-113
Publisher ID: bm-26-103
DOI: 10.11613/BM.2016.011
The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform
Markéta Škereňová[1]
Veronika Mikulová[1]
Otakar Čapoun[2]
Tomáš Zima[1]
[1] Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and First Faculty of Medicine, Charles University in Prague, Czech Republic
[2] Department of Urology, General University Hospital and First Faculty of Medicine, Charles University in Prague, Czech Republic
Author notes:
Corresponding author: marketa.jancikova@vfn.cz
Introduction
Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing.
Materials and methods
A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination.
Results
The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample.
Conclusions
The characteristics established in our study are in concordance with the manufacturer’s specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.
Keywords: lab-on-a-chip devices; capillary electrophoresis; multiplex PCR; circulating tumour cells; Agilent DNA 1000 kit
