Wang Fangfang, Wang Hui, Zhang Pengbo, Su Fengxia, Wang Honghong, Li Zhengping
Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, PR China.
Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, PR China.
Anal Chim Acta. 2021 Apr 1;1152:338281. doi: 10.1016/j.aca.2021.338281. Epub 2021 Feb 4.
MicroRNAs (miRNAs) are a class of regulatory small RNA molecules that play critical roles in a wide variety of biological processes. Abnormally expressed miRNAs have been increasingly utilized as biomarkers for cancer diagnosis. Generally, a specific cancer is associated with expression alterations of several species of miRNAs and different types of cancers are related to different miRNA species. Therefore, a universal method for multiplexed detection of miRNA targets of interest is now desirable for cancer diagnosis. In this paper, by adding an enzymatic digestion step to reduce the nonspecific adaptor dimers, we firstly improved the method to construct cDNA library of all miRNAs, which greatly increased the cDNA yield. By specifically designing DNA probes to hybridize with the cDNAs at key positions and doubly encoding DNA probes with different lengths and different fluorophores during single-base extension, each miRNA could produce a unique product, which could be separated and detected by capillary electrophoresis. Thus, miRNA targets of interest could be simultaneously detected with great specificity at single-base resolution. By using seventeen randomly selected miRNAs as the model, as low as 1.0 fM of each miRNA target could be simultaneously determined. Furthermore, we had achieved accurate analysis of multiple miRNAs in real biological RNA samples and found that several miRNAs expressed differently between cancer cells and normal cells, indicating that the proposed method had the ability to pick out aberrant expression miRNAs in real biological samples. Compared with high-throughput sequencing methods, the proposed method is simpler and specific, and very suitable for the detection of specific miRNAs associated with a disease, which shows great potential for cancer diagnosis.
微小RNA（miRNA）是一类调控性小RNA分子，在多种生物学过程中发挥关键作用。异常表达的miRNA越来越多地被用作癌症诊断的生物标志物。一般来说，特定的癌症与几种miRNA的表达改变相关，不同类型的癌症与不同的miRNA种类相关。因此，现在需要一种通用的方法来多重检测感兴趣的miRNA靶标用于癌症诊断。在本文中，通过添加酶切步骤以减少非特异性接头二聚体，我们首先改进了构建所有miRNA cDNA文库的方法，这大大提高了cDNA产量。通过专门设计DNA探针在关键位置与cDNA杂交，并在单碱基延伸过程中用不同长度和不同荧光团对DNA探针进行双重编码，每个miRNA都可以产生独特的产物，该产物可通过毛细管电泳进行分离和检测。因此，可以在单碱基分辨率下以高特异性同时检测感兴趣的miRNA靶标。以17个随机选择的miRNA作为模型，每个miRNA靶标低至1.0 fM都可以同时测定。此外，我们已经在真实的生物RNA样本中实现了对多种miRNA的准确分析，发现癌细胞和正常细胞之间有几种miRNA表达不同，这表明所提出的方法有能力在真实生物样本中挑选出异常表达的miRNA。与高通量测序方法相比，所提出的方法更简单、更具特异性，非常适合检测与疾病相关的特定miRNA，在癌症诊断中显示出巨大潜力。
