Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
Department of Electrical & Computer Engineering, University of Toronto, Toronto, ON, Canada.
Nat Chem. 2018 May;10(5):489-495. doi: 10.1038/s41557-018-0025-8. Epub 2018 Apr 2.
Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis-single-cell mRNA cytometry-that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.
细胞间基因表达的变化产生了对能够在单个细胞水平上描述表达的技术的需求。对于罕见的循环肿瘤细胞来说，这一点尤其正确，因为对其进行亚型分析和耐药性分析是非常重要的。在这里，我们描述了一种用于细胞分析的单细胞 mRNA 细胞计量术，该方法能够根据目标 mRNA 序列从全血中分离出罕见的细胞。该方法使用两类标记的磁性颗粒，以选择性地与目标 mRNA 的不同区域杂交。杂交导致形成大的磁性簇，这些磁性簇仍然局限于感兴趣的细胞内，从而能够进行磁性分离。针对特定的细胞内 mRNA 能够区分循环肿瘤细胞与正常造血细胞。不需要聚合酶链反应扩增来确定单个细胞水平的 RNA 表达水平和基因型，并且需要最小的细胞操作。为了证明这种方法，我们使用单细胞 mRNA 细胞计量术来检测前列腺癌标本中具有临床重要意义的序列。
