Department of Functional Biology (Genetic Area) and Oncology University Institute from Principado de Asturias (IUOPA), University of Oviedo, C/ Julián Clavería s/n, 33006, Oviedo, Spain; Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/ Julian Clavería 8, 33006, Oviedo, Spain.
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/ Julian Clavería 8, 33006, Oviedo, Spain.
Anal Chim Acta. 2018 Sep 6;1023:64-73. doi: 10.1016/j.aca.2018.03.047. Epub 2018 Mar 29.
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.
在过去的几年中，多重实时或定量聚合酶链反应 (PCR) 已成为用于多重基因表达变化和基因拷贝数变异 (CNV) 分析的首选方法。然而，这种测定需要使用不同的荧光标记物来标记不同的扩增序列，这大大增加了分析的成本，并限制了该技术在单个反应中同时扩增多个感兴趣的靶标的适用性。在这方面，使用凝胶电泳 (GE) 分离与电感耦合等离子体质谱 (ICP-MS) 检测相结合的方法允许通过监测 DNA 骨架中的 P 来进行无标记的多重 PCR 扩增序列 (扩增子) 的测定。通过在最佳和受控的多重 PCR 条件下，分离的扩增子的峰面积与原始样品中 DNA 模板的量成正比，从而获得定量维度。此外，通过使用 DNA 梯校准 GE-ICP-MS 系统，可以直接估计 PCR 产物的大小 (bp)。通过评估两种不同情况来评估所提出的多重策略的适用性：人卵巢癌细胞中的 CNV 和基因表达变化的测定。在第一种情况下，对 OVCAR-3 细胞中获得的 DNA 进行的四个基因 (HER2、CCNE1、GSTM1、ACTB) 的 CNV 同时定量的结果与文献数据一致，也与常规单重 qPCR 获得的结果一致。在第二种情况下，使用 ACTB 作为组成型基因，对 A2780cis 相对于 A2780 细胞中 BAX、ERCC1 和 CTR1 基因的多重基因表达变化进行测定，对顺铂敏感和耐药的细胞分别提供了与单重反应逆转录 (RT)-qPCR 相同的信息。
