Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.
Department of Radiation Oncology, Sunnybrook-Odette Cancer Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.
Anal Chem. 2020 Oct 20;92(20):14251-14258. doi: 10.1021/acs.analchem.0c03605. Epub 2020 Oct 2.
Thousands of putative microRNA (miRNA)-based cancer biomarkers have been reported, but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method that is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. The capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard), but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here, we explain the challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly negatively charged PNA-miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA-miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for the simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers.
已经报道了数千种假定的基于 microRNA（miRNA）的癌症生物标志物，但没有一种经过食品和药物管理局的验证批准。造成这种惊人差异的原因之一是缺乏一种足够强大的方法来进行验证研究，这种方法可能需要在多个机构中对数百名患者的样本进行分析，并将结果汇集在一起。基于毛细管电泳（CE）的杂交分析被证明比逆转录聚合酶链反应（当前的标准）更稳健，但它的定量下限（LOQ）超过 10 pM，而细胞裂解物中的 miRNA 浓度低于 1 pM。因此，基于 CE 的分离必须先进行柱上样品预浓缩。在这里，我们解释了基于 CE 的 miRNA 分析中样品预浓缩的挑战，并介绍了一种预浓缩方法，该方法可适用于利用肽核酸（PNA）杂交探针进行基于 CE 的 miRNA 分析。该方法结合了场放大样品堆积（FASS）和等速电泳（ITP）。我们证明 FASS-ITP 可以保留和浓缩带负电荷的近中性 PNA 与带高度负电荷的 PNA-miRNA 杂交物。我们证明，FASS-ITP 预浓缩可以与 CE 分离未反应的 PNA 探针与 PNA-miRNA 杂交物相结合，并通过 140 倍的因子提高 LOQ，降低至 0.1 pM。最后，我们将 FASS-ITP-CE 应用于粗细胞裂解物中两种 miRNA 的同时检测，并证明该方法在复杂生物基质中具有稳健性。LOQ 提高 140 倍，对生物基质的稳健性将极大地扩展基于 CE 的 miRNA 分析的适用性，使其更接近成为 miRNA 生物标志物验证的实用工具。
