液滴微流控单细胞转录组检测技术的建立和优化

doi:10.3969/j.issn.1004-616x.2019.01.002

癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (1): 9-14,21.doi: 10.3969/j.issn.1004-616x.2019.01.002

液滴微流控单细胞转录组检测技术的建立和优化

周鲁林¹, 杨润坤², 张文³, 冯林¹, 肖汀¹, 程书钧¹, 张开泰¹, 张蕾⁴

1. 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室, 癌发生及预防分子机理北京市重点实验室, 北京 100021;
2. 哈尔滨医科大学附属第二医院结直肠肿瘤外科, 哈尔滨 150086;
3. 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院免疫学研究室, 北京 100021;
4. 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院内镜科, 北京 100021

收稿日期:2018-11-15 修回日期:2018-12-29 出版日期:2019-01-31 发布日期:2019-01-31
通讯作者: 张蕾,E-mail:leizhang010@163.com E-mail:leizhang010@163.com
作者简介:周鲁林,E-mail:zhoululin1@126.com。
基金资助:
国家重点研发计划项目（2017YFC1308700，2017YFC1308702），中国医学科学院医学与健康科技创新工程（2017-I2M-1-005）

Establishment and optimization of droplet microfluidic single-cell transcriptome detection technology

ZHOU Lulin¹, YANG Runkun², ZHANG Wen³, FENG Lin¹, XIAO Ting¹, CHENG Shujun¹, ZHANG Kaitai¹, ZHANG Lei⁴

1. State Key Laboratory of Molecular Oncology, Beijing Key Laboratory for Carcinogenesis and Cancer Prevention, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021;
2. Department of Colorectal Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086;
3. Department of Immunology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021;
4. Department of Endoscope, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

Received:2018-11-15 Revised:2018-12-29 Online:2019-01-31 Published:2019-01-31

摘要/Abstract

摘要： 目的：建立基于液滴微流控技术的单细胞转录组检测平台，并对该平台的有效性和在肿瘤研究中的潜在应用方向进行探讨。方法：利用液滴微流控体系，实现单个细胞和单个标记微球在油包水型乳浊液微滴中的共同包裹，即实现单细胞的分离与标记。通过调节流路内各项参数获得最适单细胞标记率。以混合的人急性早幼粒白血病细胞系HL-60和小鼠黑色素瘤细胞系B16-F10为模拟样品，评价所建系统的各项技术指标。结果：在油相和水相流速分别为200和30 μL/min的条件下，细胞和微球浓度分别为250和400个/μL时，达到最适细胞标记率（11.87±3.75）%。模拟样品检测结果显示，输入细胞7.50万个，理论细胞标记率为11%时，可获得7 535个细胞的转录组数据，其中人和鼠转录组混合细胞有70个，仅占全部细胞的0.9%。利用转录组差异能够区分两类细胞，且能筛选出每类细胞的特异性表达标志物。结论：本研究建立了一个基于液滴微流控的单细胞转录组分析平台，平台的各项指标能够满足肿瘤学实验室要求，在肿瘤研究中具有广泛的潜在应用价值。

关键词: 单细胞测序, 微流控, 转录组, 肿瘤细胞

Abstract: OBJECTIVE:To establish and to validate a droplet microfluidic single-cell transcriptome detection platform for cancer research. METHODS:Using a droplet microfluidic system,a single cell and a single barcoded microparticle were wrapped together in an oil-water emulsion droplet. The mixed human acute promyelocytic leukemia cell line HL-60 and the mouse melanoma cell line B16-F10 were used as simulative sample to evaluate the technical indexes of the system. RESULTS:When the oil and the water phase flow rates were 200 and 30 μL/min,respectively,and the cell and microparticle concentrations were at 250 and 400/μL,the optimum cell labeling rate was reached at (11.87±3.75)%. With a simulative sample which contained 75 thousand cells and the theoretical cell labeling rate of 11%,the transcriptome data of 7 535 cells was obtained,among which there were 70 mixed human and mouse transcriptome cells accounting for only 0.9% of all cells. Transcriptome differences were used to distinguish the two types of cells and to screen out the specific expression markers among them. CONLUSION:In this study,a droplet microfluidic single-cell transcriptome detection platform was initiated and validated. The platform meets the requirements of oncology laboratory and shows extensive potential for tumor research.

Key words: single-cell sequencing, microfluidic, transcriptome, tumor cells

中图分类号:

R-331

周鲁林, 杨润坤, 张文, 冯林, 肖汀, 程书钧, 张开泰, 张蕾. 液滴微流控单细胞转录组检测技术的建立和优化[J]. 癌变·畸变·突变, 2019, 31(1): 9-14,21.

ZHOU Lulin, YANG Runkun, ZHANG Wen, FENG Lin, XIAO Ting, CHENG Shujun, ZHANG Kaitai, ZHANG Lei. Establishment and optimization of droplet microfluidic single-cell transcriptome detection technology[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(1): 9-14,21.

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液滴微流控单细胞转录组检测技术的建立和优化

Establishment and optimization of droplet microfluidic single-cell transcriptome detection technology

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