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Protocol for Microfluidic System to Automate the Preparation and Fractionation of the Nucleic Acids in the Cytoplasm Versus Nuclei of Single Cells
自动制备和分级分离单细胞细胞质与细胞核中核酸的微流体系统方法   

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Abstract

This protocol describes the extraction, fractionation, and recovery of cytoplasmic nucleic acids (e.g., cytoplasmic RNA) versus nucleic acids in the cell nucleus (including genomic DNA, gDNA) from single cells with a microfluidic system. The method enables independent, sequence-specific analyses of these critical markers (Kuriyama et al., 2015). The system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in less than 5 min, including lysis, purification, fractionation, and delivery to two output reservoirs: One for the nucleus (including gDNA and nuclear RNA) and one for cytoplasmic RNA. Each reservoir then contains high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus nucleic acids in nucleus. As described here, our protocol focuses on the analysis of cytoplasmic RNA versus gDNA from the nucleus. We have tested this protocol with mouse and human cells but not with walled cells such as plant cells.

Keywords: Single cell analysis(单细胞分析), RNA(RNA), DNA(DNA), Electrophoresis(电泳), Microfluidics(微流控芯片)

Materials and Reagents

  1. Microfluidic chip fabrication
    Fabricate a microfluidic device (Figure 1) from polydimethylsiloxane (PDMS) (Dow Corning, Sylgard® 184 Silicone Elastomer Kit) and glass slides by using soft lithography. The nominal channel width and depth of the microfluidic device are 90 μm and 35 μm, respectively. The basic design is similar to the microchannel used by Shintaku et al. (2014) except for a downstream branch channel. We have made available the CAD file for the device in the supporting information.


    Figure 1. Schematic of channel geometry. A. The letters (a to f) in the figure identify the various channel sections. Vi (for I = 1 to 5) refer to the voltages applied to platinum electrodes placed in respective reservoirs. The numbers also refer to the reservoirs, e.g., 1 = I, 2 = nNA, 3 = cNA, 4 = B, and 5 = V. B. The lengths of channel sections in the chip.

  2. Reagents for extraction
    Prepare following solutions in UltraPure DNase-/RNase-free deionized (DI) water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977 ). Prepare buffer solutions under DNA/RNA free and DNase/RNase free environment (e.g., clean room conditions)
    1. Cells (A20, mouse B cell lymphoma)
    2. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
    3. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
    4. Tris (Sigma-Aldrich, catalog number: 93362 )
    5. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
    6. Polyvinylpyrrolidone (PVP) (Sigma-Aldrich, catalog number: 437190 )
    7. HEPES (Sigma-Aldrich, catalog number: PHG0001 )
    8. Sucrose (Sigma-Aldrich, catalog number: S0389 )
    9. Leading electrolyte (LE) (see Recipes)
    10. Trailing electrolyte (TE) (see Recipes)
    11. Cell suspension buffer (see Recipes)

  3. Reagents for RT-qPCR and qPCR
    The reagents described here are for an experiment similar to Kuriyama et al. (2015) wherein we performed quantitation of gDNA for the nucleus and cytoplasmic RNA for the cytoplasm. However, we note the general lysing, fractionation, and preparation protocol described here is well applicable to other studies (e.g., analysis of nuclear versus cytoplasmic fractions of RNA from single cells).
    1. TaqMan® RT-PCR mix (2x)*
    2. TaqMan® gene expression assay
    3. TaqMan® RT enzyme mix (40x)*
    4. TaqMan® RNA-to-CTTM 1-step kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4392653 )
    5. Primer set (forward primer, reverse primer) (TaqMan® probe)
    6. DI water
    7. RT-qPCR master mix for cytoplasmic RNA analysis for two reactions (see Recipes)
    8. qPCR master mix for gDNA analysis for one reaction (see Recipes)
      *Note: TaqMan® RNA-to-CTTM 1-step kit contains both reagents.

Equipment

  1. Microscope
    A phase contrast microscope fitted with a CCD camera enables monitoring the lysing and cytoplasmic nucleic acid fraction focusing via isotachophoresis (ITP) [see on-line movies by Garcia-Schwarz et al. (2012) for a typical on-chip ITP experiment.]. It also helps monitor migration of nucleus and its fractionation from the cytoplasmic fraction in the ITP zone. Avoid using fluorescence-based visualization, e.g., intercalation dye, which may interfere specificity and stringency of downstream assay.
  2. High voltage sequencer
    A voltage sequencer (e.g., LabSmith, Inc., model: HVS448-3000D) automates the lysis, extraction, and fractionation via ITP.
    Note: A current measurement helps monitor the ITP zone migration and fractionation of the nucleus.
    1. Send the voltage sequence to the voltage sequencer.



    2. Connect the voltage outputs to platinum wire electrodes that will be placed to the reservoirs.

Procedure

  1. Filling and preparation of microfluidic system
    Each experiment should start with a new microfluidic system to avoid contaminating the sample. The washing protocol further decontaminates the microfluidic system and the associated platinum wire electrodes. Precondition the microchannel by filling reservoirs cytoplasmic nucleic acids out (cNA), nuclear nucleic acids out (nNA), and vacuum (V) with washing solutions (see below) and applying vacuum at reservoirs input (I) and branch (B) using a Y-connection and a single vacuum line. The washing solutions and the process are as follows (Video 1):
    1. 1 M NaOH with 0.1% Triton X-100 for 1 min. Wash platinum wire electrodes by dipping them into the same solution.
    2. 1 M HCl with 0.1% Triton X-100 for 1 min. Wash platinum wire electrodes by dipping them into the same solution.
    3. Deionized water with 0.1% Triton X-100 for 1 min. Wash platinum wire electrodes by dipping them into the same solution.
    4. Dry the microchannel by applying vacuum to reservoirs I and V for 1 min.
    5. Load LE to reservoirs cNA, nNA and V, and apply vacuum to reservoirs I and B for approximately 1 min.

      Video 1. Filling and preparation of microfluidic system

Recipes

  1. Leading electrolyte (LE) (pH 8.1)
    50 mM Tris
    25 mM HCl
    0.4% PVP
  2. Trailing electrolyte (TE) (pH 8.3)
    50 mM Tris
    25 mM HEPES
    0.4% PVP
  3. Cell suspension buffer (pH 8.3)
    50 mM Tris
    25 mM HEPES
    225 mM sucrose
    ~5 cells/μl cells
  4. RT-qPCR master mix for cytoplasmic RNA analysis for two reactions
    20.0 μl TaqMan® RT-PCR mix (2x)*
    2.0 μl TaqMan® gene expression assay
    1.0 μl TaqMan® RT enzyme mix (40x)*
    *Note: TaqMan® RNA-to-CTTM 1-step kit contains both reagents.
  5. qPCR master mix for gDNA analysis for one reaction
    10.0 μl TaqMan® RT-PCR mix (2x)
    1.0 μl primer set (forward primer, reverse primer, and TaqMan® probe)*
    8.0 μl DI water
    *Note: Off-the-shelf primer sets can be available from TaqMan® copy number assays.

Acknowledgments

We gratefully acknowledge funding from the National Science Foundation under CBET-1159092. H. S. acknowledges funding from Japan Society for the Promotion of Science under 22686021, 26289035, and 26630052. H. S. was supported by fellowships from the John Mung Program of Kyoto University and Marubun Research Promotion Foundation, Japan.

References

  1. Garcia-Schwarz, G., Rogacs, A., Bahga, S. S. and Santiago, J. G. (2012). On-chip isotachophoresis for separation of ions and purification of nucleic acids. J Vis Exp (61): e3890.
  2. Kuriyama, K., Shintaku, H. and Santiago, J. G. (2015). Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells. Electrophoresis 36(14): 1658-1662.
  3. Shintaku, H., Nishikii, H., Marshall, L. A., Kotera, H. and Santiago, J. G. (2014). On-chip separation and analysis of RNA and DNA from single cells. Anal Chem 86(4): 1953-1957.

简介

This protocol describes the extraction, fractionation, and recovery of cytoplasmic nucleic acids (e.g., cytoplasmic RNA) versus nucleic acids in the cell nucleus (including genomic DNA, gDNA) from single cells with a microfluidic system. The method enables independent, sequence-specific analyses of these critical markers (Kuriyama et al., 2015). The system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in less than 5 min, including lysis, purification, fractionation, and delivery to two output reservoirs: One for the nucleus (including gDNA and nuclear RNA) and one for cytoplasmic RNA. Each reservoir then contains high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus nucleic acids in nucleus. As described here, our protocol focuses on the analysis of cytoplasmic RNA versus gDNA from the nucleus. We have tested this protocol with mouse and human cells but not with walled cells such as plant cells.

关键字:单细胞分析, RNA, DNA, 电泳, 微流控芯片

材料和试剂

  1. 微流控芯片制造
    使用软光刻法由聚二甲基硅氧烷(PDMS)(Dow Corning,Sylgard 184 Silicone Elastomer Kit)和载玻片制造微流体装置(图1)。微流体装置的标称通道宽度和深度分别为90μm和35μm。除了下游分支通道之外,基本设计类似于Shintaku等人(2014)使用的微通道。我们已经在支持信息中提供了设备的CAD文件。


    图1.通道几何结构示意图。图中的字母(a到f)标识各个通道部分。 Vi(对于I = 1至5)是指施加到放置在各个储存器中的铂电极的电压。数字还指储层。例如,1 = 1,2 = nNA,3 = cNA,4 = B和5 = V.B B.。芯片中的通道部分的长度。

  2. 提取用试剂
    在UltraPure DNase-/RNase-free去离子(DI)水(Thermo Fisher Scientific,Invitrogen,目录号:10977)中制备以下溶液。在无DNA/RNA和无DNA酶/无RNA酶环境下(例如,无尘室条件下)制备缓冲液。
    1. 细胞(A20,小鼠B细胞淋巴瘤)
    2. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
    3. TritonX-100(Sigma-Aldrich,目录号:X100)
    4. Tris(Sigma-Aldrich,目录号:93362)
    5. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
    6. 聚乙烯吡咯烷酮(PVP)(Sigma-Aldrich,目录号:437190)
    7. HEPES(Sigma-Aldrich,目录号:PHG0001)
    8. 蔗糖(Sigma-Aldrich,目录号:SO389)
    9. 前导电解质(LE)(参见配方)
    10. 拖尾电解液(TE)(参见配方)
    11. 细胞悬浮缓冲液(参见配方)

  3. RT-qPCR和qPCR的试剂
    这里描述的试剂用于类似于Kuriyama等人(2015)的实验,其中我们对细胞质的核和细胞质RNA的gDNA进行定量。 然而,我们注意到,本文所述的一般裂解,分馏和制备方案适用于其它研究(例如,来自单细胞的RNA的核对细胞质级分的分析)。
    1. TaqMan RT-PCR混合物(2x)*
    2. TaqMan 基因表达测定
    3. TaqMan RT酶混合物(40x)*
    4. (Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4392653)
    5. 引物组(正向引物,反向引物)(TaqMan 探针)
    6. 去离子水
    7. RT-qPCR主混合物用于两个反应的细胞质RNA分析(参见配方)
    8. qPCR主混合物用于一次反应的gDNA分析(参见配方)
      *注意:TaqMan /sup> 单步试剂盒含有两种试剂。

设备

  1. 显微镜
    装配有CCD照相机的相差显微镜能够通过等速电泳(ITP)监测聚焦的裂解和细胞质核酸级分[参见Garcia-Schwarz等人的在线电影(2012)片上ITP实验。它还有助于监测核的迁移及其从ITP区中的胞质部分的分馏。避免使用基于荧光的可视化,例如插入染料,这可能会干扰下游测定的特异性和严格性。
  2. 高压定序器
    电压定序器(例如LabSmith,Inc.,型号:HVS448-3000D)通过ITP自动裂解,提取和分级分离。
    注意:当前的测量有助于监测ITP区迁移和细胞核的分馏。
    1. 将电压序列发送到电压定序器。



    2. 将电压输出连接到将放置到储存器的铂丝电极

程序

  1. 微流体系统的填充和制备
    每个实验应该从一个新的微流体系统开始,以避免污染样品。 洗涤方案进一步净化微流体系统和相关的铂丝电极。 通过用洗涤溶液(见下文)填充储库细胞质核酸外(cNA),核核酸外(nNA)和真空(V)并在储槽输入(I)和分支(B) Y连接和单个真空管线。 洗涤溶液和过程如下(视频1):
    1. 1M NaOH与0.1%Triton X-100一起温育1分钟。 将铂丝电极浸入同一溶液中清洗铂丝电极。
    2. 1M HCl和0.1%Triton X-100洗脱1分钟。将铂丝电极浸入同一溶液中清洗铂丝电极。
    3. 用0.1%Triton X-100去离子水1分钟。将铂丝电极浸入同一溶液中清洗铂丝电极。
    4. 通过对储层I和V施加真空1分钟来干燥微通道。
    5. 加载LE到油藏cNA,nNA和V,并对油藏I和B应用真空约1分钟。

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  2. 加载单细胞和提取(视频2)
    这里描述的协议终于与20微升细胞质RNA样本。 使用括号中的体积也可获得10μl细胞质RNA样品的方案。
    1. 从所有容器中除去残余溶液,并向容器B中加入20μl(10μl)LE至容器cNA,nNA和V,以及20μl(10μl)TE。
    2. 加载2微升(1微升)细胞悬浮液(约5细胞/微升)进入库I.从这个低浓度溶液引入单个细胞通过应用真空到库V.
    3. 目视确认在通道的区域a中,在储存器I和交叉接合处之间的隔离的单个细胞,并将20μl(10μl)TE缓冲液添加到储存器I中。
    4. 将铂丝电极放入储层I,B,cNA和nNA,并启动裂解,提取和分馏的电压序列。
    5. 用1μl微量移液管从DNA Out中提取细胞核,转移到含有19μlqPCR主混合物的PCR管中。
    6. 从RNA Out中提取20μl(10μl)RNA样品,转移到PCR管中
    7. 将9μlRNA样品与11μlRT-qPCR主混合物混合
    8. 用20μl总测定体积进行RT-qPCR和qPCR分析
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食谱

  1. 前导电解质(LE)(pH 8.1)
    50 mM Tris
    25 mM HCl
    0.4%PVP
  2. 拖尾电解液(TE)(pH 8.3)
    50 mM Tris
    25 mM HEPES
    0.4%PVP
  3. 细胞悬浮缓冲液(pH 8.3)
    50 mM Tris
    25 mM HEPES
    225mM蔗糖 〜5个细胞/μl细胞
  4. RT-qPCR主混合物用于两次反应的细胞质RNA分析 20.0μlTaqMan RT-PCR混合物(2x) *
    2.0μlTaqMan 基因表达测定
    1.0μlTaqMan RT酶混合物(40x) *
    注意:TaqMan > TM 一步试剂盒含有两种试剂。
  5. qPCR主混合物用于一次反应的gDNA分析
    10.0μlTaqMan RT-PCR混合物(2x)
    1.0μl引物组(正向引物,反向引物和TaqMan 探针) *
    8.0μl去离子水
    注意:现有的引物组可以从TaqMan > 拷贝数测定。

致谢

我们衷心感谢国家科学基金会在CBET-1159092下的资助。 H. S.承认来自日本促进科学协会的资助22686021,26289035和26630052. H. S.得到京都大学约翰门方案和日本Marubun研究促进基金的研究金的支持。

参考文献

  1. Garcia-Schwarz,G.,Rogacs,A.,Bahga,SS和Santiago,JG(2012)。  芯片等速电泳用于分离离子和纯化核酸。

    (61):e3890。
  2. Kuriyama,K.,Shintaku,H。和Santiago,JG(2015)。  Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells. 36(14):1658-1662。
  3. Shintaku,H.,Nishikii,H.,Marshall,LA,Kotera,H。和Santiago,JG(2014)。  从单个细胞的芯片上分离和分析RNA和DNA。 Anal Chem 86(4):1953-1957。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kuriyama, K., Shintaku, H. and Santiago, J. G. (2016). Protocol for Microfluidic System to Automate the Preparation and Fractionation of the Nucleic Acids in the Cytoplasm Versus Nuclei of Single Cells. Bio-protocol 6(12): e1844. DOI: 10.21769/BioProtoc.1844.
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