Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States.
Anal Chem. 2024 Jan 23;96(3):1371-1379. doi: 10.1021/acs.analchem.3c05213. Epub 2024 Jan 6.
Proteins and microRNAs (miRNAs) act in tandem within biological pathways to regulate cellular functions, and their misregulation has been correlated to numerous diseases. Because of their interconnectedness, both miRNAs and proteins must be evaluated together to obtain accurate insights into the molecular pathways of pathogenesis. However, few analytical techniques can measure both classes of biomolecules in parallel from a single biological sample. Here, microfluidic digital quantitative PCR (dqPCR) was developed to simultaneously quantify miRNA and protein targets in a multiplexed assay using a single detection chemistry. This streamlined analysis was achieved by integrating base-stacking PCR and immuno-PCR in a microfluidic array platform. Analyses of let-7a (miRNA) and IL-6 (protein) were first optimized separately to identify thermocycling and capture conditions amenable to both biomolecules. Singleplex dqPCR studies exhibited the expected digital signals and quantification cycles for both analytes over a range of concentrations. Multiplexed analyses were then conducted to quantify both let-7a and IL-6 with high sensitivity (LODs ∼ 3 fM) over a broad dynamic range (5-5000 fM) using only standard PCR reagents. This multiplexed dqPCR was then translated to the analysis of HEK293 cell lysate, where endogenous let-7a and IL-6 were measured simultaneously without sample purification or pretreatment. Collectively, these studies demonstrate that the integration of BS-PCR and immuno-PCR achieves a sensitive and streamlined approach for multiplexed analyses of miRNAs and proteins, which will enable researchers to gain better insights into disease pathogenesis in future applications.
蛋白质和 microRNAs (miRNAs) 在生物途径中协同作用,调节细胞功能,它们的失调与许多疾病有关。由于它们的相互关联性,miRNAs 和蛋白质都必须一起评估,才能准确了解发病机制的分子途径。然而,很少有分析技术可以从单个生物样本中同时平行测量这两类生物分子。在这里,微流控数字定量 PCR (dqPCR) 被开发出来,用于使用单一检测化学物质在多重测定中同时定量 miRNA 和蛋白质靶标。这种简化的分析是通过将碱基堆积 PCR 和免疫-PCR 集成到微流控阵列平台中实现的。首先分别对 let-7a(miRNA)和 IL-6(蛋白质)进行了分析优化,以确定适用于两种生物分子的热循环和捕获条件。单重 dqPCR 研究显示,在一系列浓度范围内,两种分析物均表现出预期的数字信号和定量循环。然后进行了多重分析,使用标准 PCR 试剂即可在较宽的动态范围内(5-5000 fM)以高灵敏度(LOD∼3 fM)定量 let-7a 和 IL-6。然后将这种多重 dqPCR 转化为 HEK293 细胞裂解物的分析,其中无需样品纯化或预处理即可同时测量内源性 let-7a 和 IL-6。总之,这些研究表明,BS-PCR 和免疫-PCR 的集成实现了 miRNA 和蛋白质的敏感和简化的多重分析方法,这将使研究人员能够在未来的应用中更好地了解疾病的发病机制。