Multiplex polymerase chain reaction in combination with gel electrophoresis-inductively coupled plasma mass spectrometry: A powerful tool for the determination of gene copy number variations and gene expression changes

During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for t...

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Bibliographic Details
Published in:Analytica chimica acta 2018-09, Vol.1023, p.64-73
Main Authors: Fernández Asensio, A., Iglesias, T., Cotarelo, A., Espina, M., Blanco-González, E., Sierra, L.M., Montes-Bayón, M.
Format: Article
Language:English
Subjects:
Amplification
Calibration
Cancer
Cells
Cisplatin
Copy number
Copy number variations
Cost analysis
Deoxyribonucleic acid
DNA
Electrophoresis
Emission spectroscopy
ErbB-2 protein
ERCC1 protein
Fluorescence
Gel electrophoresis
Gel electrophoresis coupled to inductively coupled plasma mass spectrometry
Gene expression
Gene expression analysis
Gene sequencing
Genes
GSTM1 protein
Inductively coupled plasma mass spectrometry
Mass spectrometry
Mass spectroscopy
Multiplex polymerase chain reaction
Multiplexing
Nucleotide sequence
Ovarian cancer
Polymerase chain reaction
Quantitation
Reverse transcription
Reverse transcription-quantitative polymerase chain reaction
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Summary:During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for the different amplified sequences, which increases significantly the costs of the analysis and limits the applicability of the technique for simultaneous amplification of many targets of interest in a single reaction. In this regard, the use of the coupling between gel electrophoresis (GE) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection allows the label-free determination of multiplex PCR-amplified sequences (amplicons) by monitoring the P present in the DNA backbone. The quantitative dimension is obtained since under optimal and controlled multiplex PCR conditions the peak areas of the separated amplicons are directly proportional to the amount of DNA template in the original sample. Moreover, the calibration of the GE-ICP-MS system with a DNA ladder permits direct estimation of the size (bp) of the PCR products. The suitability of the proposed multiplex strategy has been evaluated addressing two different situations: determination of CNVs and gene expression changes in human ovarian cancer cells. In the first case, the results obtained for the simultaneous quantitation of CNVs of four genes (HER2, CCNE1, GSTM1, ACTB) on DNA obtained from OVCAR-3 cells were in accordance with the literature data, and also with the results obtained by conventional simplex qPCR. In the second case, multiplex gene expression changes of BAX, ERCC1 and CTR1 genes, using ACTB as constitutive gene, on A2780cis respect to A2780 cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR. [Display omitted] •Conventional multiplex PCR was combined with GE-ICP-MS for CNV and gene expression analyses in cancer cells.•Amplicons were separated, identified and label-free quantitate through 31P monitoring.•CNVs results obtained with this method were in accordance to literature data and to uniplex qPCR results.•This method provided the same information on relative gene expression for three genes, as single reaction RT-qPCR.
ISSN:0003-2670
1873-4324
DOI:10.1016/j.aca.2018.03.047