Cohen Limor, Hartman Mark R, Amardey-Wellington Aaron, Walt David R
Department of Chemistry, Tufts University, Medford, MA 02155, USA.
Nucleic Acids Res. 2017 Aug 21;45(14):e137. doi: 10.1093/nar/gkx542.
MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.
微小RNA（miRNA）参与许多生物学途径，准确检测miRNA对于诊断包括癌症在内的多种疾病至关重要。然而，当前大多数miRNA检测方法需要冗长的样品制备和扩增步骤，这可能会使结果产生偏差。此外，缺乏特异性和可重复性给生物样品中循环miRNA的检测带来了各种挑战。在这项工作中，我们应用单分子阵列（Simoa）技术开发了一种超灵敏夹心测定法，用于直接检测多种miRNA而无需预扩增。我们成功检测到了飞摩尔浓度的miRNA（检测限[LOD]范围为1至30 fM），并且具有高特异性（能够区分单核苷酸错配的miRNA）。该方法对一系列不同的靶序列均有效，表明这是一种通用的miRNA检测方法。为了证明该技术的实际应用，我们在包括人血清和总RNA在内的多种样品类型中检测了miRNA。Simoa方法的高灵敏度和简单工作流程为基于miRNA的人类疾病诊断带来了优异的优势。
