Baptista Pedro Viana
Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, UCIBIO, Universidade Nova de Lisboa, Caparica, Portugal.
Methods Mol Biol. 2020;2118:251-268. doi: 10.1007/978-1-0716-0319-2_19.
Nanotechnology provides new tools for gene expression analysis that allow for sensitive and specific characterization of prognostic signatures related to cancer. Cancer is a complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus allows for a more accurate indication into the degree of cancerous activity than either locus alone. Metal nanoparticles have been widely used as labels for in vitro identification and quantification of target sequences.Here we describe the synthesis of nanoparticles with different noble metal compositions in an alloy format that are then functionalized with thiol-modified ssDNA (nanoprobes). We also show how such nanoprobes are used in a non-cross-linking colorimetric method for the direct detection and quantification of specific mRNA targets, without the need for enzymatic amplification or reverse-transcription steps. The different metals in the alloy provide for distinct absorption spectra due to their characteristic plasmon resonance peaks. The color multiplexing allows for simultaneous identification of different mRNA targets involved in cancer development. A comparison of the absorption spectra of the nanoprobe mixtures taken before and after induced aggregation of metal nanoparticles allows to both identify and quantify each mRNA target. We describe the use of gold and gold-silver alloy nanoprobes for the development of the non-cross-linking method to detect a specific BCR-ABL fusion gene (e.g., e1a2 and e14a2) mRNA target associated with chronic myeloid leukemia (CML) using 10 ng/μL of unamplified total human RNA. Additionally, we demonstrate the use of this approach for the direct diagnostics of CML. This simple methodology takes less than 50 min to complete after total RNA extraction with comparable specificity and sensitivity to the more commonly used methods.
纳米技术为基因表达分析提供了新工具，可对与癌症相关的预后特征进行灵敏且特异的表征。癌症是一种复杂疾病，多个基因位点对其表型都有影响。能够同时监测源自每个位点的差异表达，相比单独监测任何一个位点，能更准确地指示癌症活动程度。金属纳米颗粒已被广泛用作体外识别和定量目标序列的标记物。在此，我们描述了以合金形式合成具有不同贵金属成分的纳米颗粒，然后用硫醇修饰的单链DNA（纳米探针）进行功能化。我们还展示了如何将这种纳米探针用于一种非交联比色法，直接检测和定量特定的mRNA靶标，无需酶促扩增或逆转录步骤。合金中的不同金属因其特征性等离子体共振峰而具有不同的吸收光谱。颜色复用允许同时识别参与癌症发展的不同mRNA靶标。比较金属纳米颗粒诱导聚集前后纳米探针混合物的吸收光谱，可识别和定量每个mRNA靶标。我们描述了使用金和金银合金纳米探针开发非交联方法，以检测与慢性粒细胞白血病（CML）相关的特定BCR-ABL融合基因（如e1a2和e14a2）mRNA靶标，使用10 ng/μL未扩增的总人RNA。此外，我们展示了这种方法用于CML的直接诊断。这种简单方法在总RNA提取后不到50分钟即可完成，其特异性和灵敏度与更常用的方法相当。
