单细胞中全转录组无偏倚扩增新方法的研究及其临床应用效果评价

Single-cell analysis plays an important role to reveal molecular mechanisms of a living system and the pathogenesis of disease. Due to technical limitations, technologies of gene expression analysis in single cells frequently cause bias and have the low detection sensitivity. In order to solve these problems, in the present study we established a non-biased global amplification method for analyzing gene expression in single cells with magnetic beads for immobilizing the single-cell cDNA library. In this method, the reverse transcription (RT) primers immobilized on beads capture mRNA and synthesized the immobilized cDNA library. Then poly(A) tailing, 2nd-strand cDNA synthesis, global amplification and sequencing were carried out. Preliminary experimental results obtained by applying this method indicated the feasibility of combining a variety of nucleic acid synthesis technology, material science, and high-throughput sequencing as a novel way in single cell analysis. By using the beads as the supporting material for immobilizing library, not only a highly efficient RT of a small amount of nucleic acid in single cells was achieved, but also inhibition of the residual solution to downstream reactions was eliminated through washing the beads. High efficiency and high fidelity of the reactions were realized. As whole transcriptomes are amplified by a pair of common primers, the method possesses non-bias, high throughput, high gene coverage, and high sensitivity. Here, we plan to further optimize the experimental parameters in various steps, thoroughly check this method by applying it in the detection of gene expression profilings of single stem cells during the differentiation process, and finally provide a novel way for the non-biased analysis of whole transcriptomes in single cells.

单细胞分析对生命活动的分子机理及疾病发生机制的研究至关重要。由于技术的限制，单细胞全基因表达谱分析技术存在定量易产生偏倚、检测灵敏度低的问题。前期工作中，我们通过磁珠固定反转录引物，高效制备单细胞固相cDNA文库，结合加尾技术及高通量扩增测序技术，创新性地建立了"基于磁性微球与全转录组无偏倚扩增技术分析单细胞全基因表达谱的方法"。预实验结果提示该方法将多种核酸合成技术、材料科学和高通量测序结合的研究思路具有可行性。该方法采用磁珠作为文库的固相介质，不但实现了单细胞中少量核酸的高效反转录，且通过洗涤磁珠消除了残留液对下游反应的干扰，实现整个检测过程高效且高保真。再使用公用引物对文库中所有片段进行同时扩增，具有定量无偏倚、高通量、基因覆盖率高及高灵敏度等优点。本研究拟进一步优化各步骤实验参数，并在诱导分化过程中单个干细胞基因表达动力学分析中进行论证，将为单细胞全基因表达谱无偏倚分析提供新方法。

疾病的发生发展和细胞状态紧密相关，单细胞是现实生命系统中最小单位，其之间的状态和交互网络日益引起人们的广泛关注，对单细胞基因表达的准确定量至关重要。单细胞研究的难点在于遗传物质少，对检测技术灵敏度要求高，易产生扩增偏倚等。本课题的研究实现单细胞微量mRNA的高效反转录，无偏倚全转录组扩增，实现单细胞基因表达量的精准分析。. 课题将单细胞固相磁珠cDNA文库技术与新一代基因测序技术相结合，建立了“基于磁性微球与全转录组无偏倚扩增技术分析单细胞全基因表达谱的方法”。磁珠固定cDNA文库法通过增加洗涤步骤去除样品中残留的试剂，下一步反应可以在最优条件下进行，没有cDNAs损失。在评估误差来源后发现，最初几个步骤是关键。相比于传统方法，本法展现出了其好的重复性、定量准确性和无偏倚性。. 一次单细胞分析所需的微球数及单个微球上固定的反转录引物量对下游反应效率及偏倚易产生影响。实验中发现，使用外切核酸酶切除磁珠上未发生反转录延伸反应的引物会产生偏倚，因此改用降低磁珠表面固定反转录引物量的方法取代使用外切核酸酶切，两步PCR反应的总扩增效率提高了30倍。. 为了降低由随机误差产生的可能偏倚，一个样本被分成四份进行加尾、第二链cDNA合成及PCR反应，最后再将四份反应产物合并分析。为了减少PCR扩增偏倚，使用两步PCR法，每步PCR仅使用低循环数，两步PCR之间加入纯化步骤，这样的改进有效地降低了反应循环数并实现了高效扩增。当使用3’端为poly(T)的UP2引物作为第二链cDNA合成引物时，扩增产物的片段覆盖范围很宽，针对该问题，课题改用了3’端为poly(T)加上VN (V 是dA, dC或dG；N是dA, dC, dG或dT)的锚定引物作为第二链cDNA合成引物，引物的3’端仅与cDNAs的5’端poly(A)的尾巴杂交，使得扩增产物的片段覆盖范围变窄，更易于高效纯化，扩增效率提高了数十倍。. 改进后的方法可将来自于单细胞的2-pg mRNA扩增放大108倍得到200 mg的cDNA文库，用于下游检测，转录子的扩增偏差小于1.5倍。该技术成功地应用于白血病人免疫细胞系单细胞基因表达量的检测。