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Capillary Electrophoresis in Hydroxyethylcellulose Solutions for the Analysis of dsDNA, dsRNA, and siRNA
毛细管电泳法分析羟乙基纤维素溶液中的dsDNA、 dsRNA 和 siRNA   

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Abstract

Capillary electrophoresis (CE) is identified as a promising technology for the study of nucleic acid molecules because of its high efficiency, high throughput with automation and integration. Compared to the traditional method of slab gel electrophoresis (SGE), the advantages of CE cannot be emphasized more. Most of CE process, including sample injection, detection and data analysis, is able to be automated which will save great labor for industrial and research labs. CE used the separation channel with micrometer-scale diameter, so the joule heat is easy to be dissipated during electrophoresis. Thus high separation voltage (> 100 V/cm) is allowed in CE while in SGE (usually ~10 V/cm) it usually causes severe band broadening. Because the band broadening is restrained efficiently in CE, it is capable of detecting minute samples and becoming more sensitive than SGE. The advantage of allowing high voltage consequently speeds up the CE separation and yields a better throughput compared to SGE. CE costs less reagents, for example buffer solutions, sieving matrix, dye reagents etc. In addition, the micrometer-scale channel is easy to be integrated with upstream and downstream sample treatment units, forming a lab on a chip. This merit of CE already attracted considerable interests among researchers from various areas.

The difficulties of CE involve filling the gels (agarose or cross-linked polyacrylamide) into the capillary tube. Also, the reproducibility and the life-time of the gel-capillary are limited. But the small-diameter capillary allows to use replaceable polymer solutions, which can efficiently prevent the convection of the separation buffer. Polymer solutions are easier to be filled into the capillary and yield more stable separations. Thus, those difficulties are resolved by doing capillary polymer electrophoresis (CPE), which is going to be described in this protocol.

Several separation modes, for example, capillary gel electrophoresis (CGE), CPE, capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and so on, have been developed for analysis of different kinds of molecules. Here, we introduce the protocol for CPE in detail, which is for the separation of dsDNA, dsRNA (including siRNA) molecules. Polymer solutions are filled into the capillary as a sieving matrix for double strand nucleic acids separation. Hydroxyethylcellulose (HEC) polymer is employed as the sieving polymer in this case. A home-built CE system is described in detail.

Materials and Reagents

  1. Micro-centrifuge or PCR tube (200 μl)
  2. Pipettes (0.1-10 μl and 10-200 μl) and the corresponding tips
  3. 0.22 μm filter membrane
  4. dsDNA analytes (Takara, catalog number: 3420A )
  5. dsRNA (including siRNA) analytes (Takara, catalog number: 3430 )
  6. Ultra-pure water, with resistance of 18.25 Ω
  7. 2-hydroxyethyl cellulose (MW: 1,300k) (Sigma-Aldrich, catalog number: 434981 )
  8. TBE (Tris-borate-EDTA) powder (Takara, catalog number: T905 )
  9. SYBR Green II RNA Gel Stain, 10,000x concentrate in DMSO (Thermo Fisher Scientific, catalog number: S-7564 )
  10. Storage 1% HEC polymer solution (see Recipes)
  11. 0.5x TBE solution (see Recipes)
  12. 100x SYBR Green II (see Recipes)

Equipment

  1. Home-built CE system (Figure 1) which includes:
    1. A coated fused silica capillary with a detection window (Polymicro, catalog number: TSP075375 )
    2. A high voltage power supplier (see Note 8)
    3. A fluorescence microscope assembled with a fluorescent excitation equipment (Equipment 1d) and a fluorescent emission collection equipment (Equipment 1e) (Olympus, model: IX73 )
    4. The fluorescent excitation equipment constituted by a mercury lamp and an optical cube, which was used to transmit light from the mercury lamp into 460-495 nm fluorescence excitation spectrum (Olympus, model: U-MWIB3 )
    5. The fluorescent emission collection equipment constituted by an optical filter and a photomultiplier tube (PMT) (Hamamatsu Photonics, catalog number: H8249-101 , for alternate H7827-001)
      Note: The optical filter transmits the emission spectrum from dye-nucleonic acid conjugate to the PMT and filters background light from the detection window.
    6. The home-built CE system including a Labview system (National Instrument, model: NI USB 6212 ) and a computer
      Note: Users are able to control the high voltage supplier, monitor fluorescent signal from the detection window and collect data from PMT by the LabVIEW software.
  2. Vacuum pump (see Note 9)
  3. Centrifuge (> 3,200 x g)
  4. Ultra-pure water system (see Note 10)


    Figure 1. Schematic diagram of the assembly of the CE system

Software

  1. LabVIEW software

Procedure

  1. Prepare CE sample. Collect the dsDNA/dsRNA analytes into a micro-centrifuge tube, for example, a 200 μl volume micro-centrifuge tube. Dissolve or dilute the dsDNA/dsRNA analytes with 0.5x TBE (see Note 3).
  2. Prepare 0.5% HEC polymer solution (see Note 4). Take one micro-centrifuge tube, pipet into 100 μl storage 1% HEC polymer solution, then add into 96 μl 0.5x TBE solution and 4 μl 100x SYBR Green II. Mix the buffer solution by pipetting for several times. Split the 0.5% HEC polymer solution in another micro-centrifuge tube. Centrifuge the micro-centrifuge tubes for 5-20 sec so that the bubbles are removed from the buffer solution. In this case, we take the 0.5% HCE for an example. The concentration of polymer solution affects the separation performance of CPE greatly (Liu et al., 2015). Users are suggested to adjust the concentration of polymer solution according to the sample to be analyzed.
  3. Clean the inner wall of the capillary (Figure 2). Flush the capillary by ultra-pure water for ~30 sec using the vacuum pump. The sample will be injected at the cathode end of the capillary. In order to avoid inappropriate sample injections, we suggest placing the vacuum pump at the anode end of the capillary.
  4. Fill the 0.5% HEC polymer solution into capillary (Figure 2) as the same way of step 3.


    Figure 2. Schematic plot of filling the 0.5% HEC polymer solution into capillary and cleaning the inner wall of the capillary

  5. Set up CE parameters on the Labview interface: sample loading voltage, sample loading time, electric field intensity. When different samples are to be analyzed, those parameters are supposed to be optimized to achieve preferable separation.
  6. Inject sample. As shown in Figure 3, immerse the cathode of power supplier and the corresponding end of the capillary into the sample solution in a micro-centrifuge tube. Immerse the anode of power supplier and the corresponding end of the capillary into a micro-centrifuge tube which contained 0.5% HEC polymer solution. Make sure the capillary is well connected with the power supplier. Through the LabVIEW control software installed on the computer, set on the high voltage power supplier. So that the high voltage is applied on the capillary and the sample is injected electrokinetically.


    Figure 3. Schematic plot of injecting sample

  7. Start CE separation. Remove the sample micro-centrifuge tube and place a micro-centrifuge tube which contained 0.5% HEC polymer solution in the same way. Make sure the capillary is well connected with the power supplier.
  8. The fluorescent signal can be monitored by the LabVIEW software during the CE separation process. In the first CE run, when the last signal is observed which is supposed from the last analyte, keep the CE process on for another same duration time in order to ensure no other signals appear. Save CE data. Proceed data analysis.
  9. Clean the inner wall of capillary following step 3. The clean-up prevents the next CPE run from contamination. If no more CPE run needed, it helps to maintain the capillary for long-term storage.

Representative data



Figure 4. Electropherogram of (a) dsRNA fragments and (b) dsDNA fragments in 1.2% HEC (MW: 1,300k) solution. Capillary electrophoresis was performed at 100V/cm. The total length and the effective length of the capillary were 9.0 cm and 6.0 cm, respectively. The sample was loaded at 100 V/cm for 2.0 sec. The dsDNA sample and dsRNA sample was diluted into 13 ng/μl and 20 ng/μl, respectively, using 0.5x TBE.

Notes

  1. Denature is not obligatory for electrophoretic separation of dsDNA and dsRNA molecules.
  2. You can keep dsDNA and dsRNA analytes at 4 °C for short-term storage, and keep them at -20 °C for long term storage.
  3. Analytes are suggested to be resolved into 0.5x TBE in order to ensure the electrokinetic injection is the same for every time.
  4. Because the polymer solutions are viscous and take a long time to get resolved homogenous, it is recommended to prepare a concentrated polymer solution for storage. Before CPE experiment, the concentrated polymer solution can be diluted to a required concentration conveniently. In this case, we demonstrated the protocol to prepare the 0.5% HEC polymer solution with a final concentration of 0.5x TBE and 2x SYBR Green II.
  5. HEC polymers in various MWs, for example 90k-1,300k, can be used for dsDNA and dsRNA analysis. If the MW of HEC polymer is low, high concentration is recommended for dsDNA and dsRNA separation.
  6. SYBR Green I can also be used for dsDNA and dsRNA dying.
  7. It is suggested to keep capillaries in ultra-pure water for storage in order to maintain the performance of coated surface.
  8. Please make sure that the ripple of high voltage should be less than 0.1% of the applied voltage, and the slew rate for high voltage should be more than 20 V/μsec. These parameters are usually written in the product specification.
  9. If the capillary is long, a high pump pressure or a syringe is recommended in order to fill viscous polymer solution into the capillary. If the capillary is short, low pressure is preferred in case bubble formed inside the capillary. In our lab, the common capillary length is no more than 20 cm. The ultimate pressure of our vacuum pump is 26.6 x 103 Pa.
  10. The ultra-pure water system is supposed to produce water which meets ASTM (American Society of Testing Materials) Type-1.1 standard.
  11. We believe that the commercial available CE system is compatible with this protocol with an accommodation of the experimental process.

Recipes

  1. Storage 1% HEC polymer solution, 10 ml
    1 g HEC powder
    9 ml 0.5x TBE
    Stir the solution at room temperature for at least 24 h in order to achieve a homogenous polymer solution. Keep storage polymer solutions at room temperature.
  2. 0.5x TBE 1,000 ml
    50 ml 10x TBE
    950 ml ultra-pure water
    Filter the 0.5x TBE solution twice using a 0.22 μm filter membrane.
    Keep at room temperature.
  3. 100x SYBR Green
    1 μl 10,000x SYBR Green
    99 μl ultra-pure water
    Keep at 2-8 °C for at least one month.

Acknowledgments

This work was adapted and modified from Yamaguchi and Liu et al., 2015.
This project was partly supported by Grand-in-Aid for Scientific Research [No. 25600049 (Y. Y.), A15H038270 (Y. Y)], JSPS, Japan. We acknowledged on partly financial support by East China University of Science and Technology (No. YK0142119).

References

  1. Li, Z., Dou, X., Ni, Y., Chen, Q., Cheng, S. and Yamaguchi, Y. (2012). Is pulsed electric field still effective for RNA separation in capillary electrophoresis? J Chromatogr A 1229: 274-279.
  2. Liu, C., Yamaguchi, Y., Zhu, X., Li, Z., Ni, Y. and Dou, X. (2015). Analysis of small interfering RNA by capillary electrophoresis in hydroxyethylcellulose solutions. Electrophoresis 36(14): 1651-1657.
  3. Sumitomo, K., Sasaki, M. and Yamaguchi, Y. (2009). Acetic acid denaturing for RNA capillary polymer electrophoresis. Electrophoresis 30(9): 1538-1543.
  4. Todorov, T. I., Yamaguchi, Y. and Morris, M. D. (2003). Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids. Anal Chem 75(8): 1837-1843.
  5. Yamaguchi, Y., Todorov, T. I., Morris, M. D. and Larson, R. G. (2004). Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions. Electrophoresis 25(7-8): 999-1006.

简介

毛细管电泳(CE)被认为是研究核酸分子的有前景的技术,因为它具有高效率,具有自动化和整合的高通量。与传统的平板凝胶电泳(SGE)方法相比,CE的优点不再强调。大多数CE过程,包括样品注入,检测和数据分析,能够自动化,这将为工业和研究实验室节省大量的劳动力。 CE使用具有微米尺度直径的分离通道,因此在电泳期间焦耳热容易被消散。因此,在CE中允许高分离电压(> 100V/cm),而在SGE(通常〜10V/cm)下,其通常引起严重的带拓宽。因为在CE中能够有效地抑制谱带展宽,所以它能够检测微小的样品并且比SGE更敏感。允许高电压的优点因此加速了CE分离并且与SGE相比产生更好的通量。 CE成本较低的试剂,例如缓冲溶液,筛分基质,染料试剂等。此外,微米级通道容易与上游和下游样品处理单元集成,在芯片上形成实验室。 CE的这个优点已经吸引了来自各个领域的研究人员的相当大的兴趣。
   CE的困难涉及将凝胶(琼脂糖或交联聚丙烯酰胺)填充到毛细管中。此外,凝胶毛细管的再现性和寿命受到限制。但是小直径毛细管允许使用可更换的聚合物溶液,其可以有效地防止分离缓冲液的对流。聚合物溶液更容易填充到毛细管中并产生更稳定的分离。因此,通过进行毛细管聚合物电泳(CPE)解决了这些困难,这将在本协议中描述。
  已经开发了几种分离模式,例如毛细管凝胶电泳(CGE),CPE,毛细管区带电泳(CZE),毛细管等速电泳(CITP)等,用于分析不同种类的分子。在这里,我们详细介绍了CPE的协议,这是用于分离dsDNA,dsRNA(包括siRNA)分子。将聚合物溶液填充到毛细管中作为用于双链核酸分离的筛分基质。在这种情况下使用羟乙基纤维素(HEC)聚合物作为筛分聚合物。详细描述了自制CE系统。

材料和试剂

  1. 微量离心机或PCR管(200μl)
  2. 移液器(0.1-10μl和10-200μl)和相应的提示
  3. 0.22μm滤膜
  4. dsDNA分析物(Takara,目录号:3420A)
  5. dsRNA(包括siRNA)分析物(Takara,目录号:3430)
  6. 超纯水,电阻为18.25欧姆
  7. 2-羟乙基纤维素(MW:1300k)(Sigma-Aldrich,目录号:434981)
  8. TBE(Tris-硼酸盐-EDTA)粉末(Takara,目录号:T905)
  9. SYBR Green II RNA凝胶染色剂,DMSO中10,000x浓缩物(Thermo Fisher Scientific,目录号:S-7564)
  10. 存储1%HEC聚合物溶液(参见配方)
  11. 0.5x TBE解决方案(参见配方)
  12. 100x SYBR Green II(请参阅配方)

设备

  1. 家用CE系统(图1),包括:
    1. 具有检测窗的涂覆的熔融石英毛细管(Polymicro,目录号:TSP075375)
    2. 高压电源(见注8)
    3. 装配有荧光激发设备(设备1d)和荧光发射收集设备(设备1e)(Olympus,型号:IX73)的荧光显微镜
    4. 由汞灯和光学立方体构成的荧光激发设备用于将来自汞灯的光传输到460-495nm荧光激发光谱(Olympus,型号:U-MWIB3)。
    5. 由滤光器和光电倍增管(PMT)(Hamamatsu Photonics,目录号:H8249-101,用于交替的H7827-001)构成的荧光发射收集设备
      注意:光学滤镜将染料 - 核酸共轭的发射光谱传输到PMT,并从检测窗口过滤出背景光。
    6. 包括Labview系统(国家仪器,型号:NI USB 6212)和计算机的本地CE系统
      注意:用户可以通过LabVIEW软件控制高压供应商,从检测窗口监测荧光信号并从PMT收集数据。
  2. 真空泵(见注9)
  3. 离心机(> 3,200 x g )
  4. 超纯水系统(见注10)


    图1. CE系统装配示意图

软件

  1. LabVIEW软件

程序

  1. 准备CE样品。将dsDNA/dsRNA分析物收集到微量离心管中,例如,200μl体积的微量离心管中。用0.5x TBE溶解或稀释dsDNA/dsRNA分析物(见注释3)。
  2. 制备0.5%HEC聚合物溶液(见注4)。取一个微量离心管,移液至100μl储存1%HEC聚合物溶液,然后加入96μl0.5x TBE溶液和4μl100x SYBR Green II。通过吸移混合缓冲液几次。在另一个微量离心管中分离0.5%HEC聚合物溶液。离心微离心管5-20秒,以便从缓冲溶液中去除气泡。在这种情况下,我们以0.5%HCE为例。聚合物溶液的浓度大大影响了CPE的分离性能(Liu等人,2015)。建议用户根据要分析的样品调整聚合物溶液的浓度
  3. 清洁毛细管的内壁(图2)。使用真空泵用超纯水冲洗毛细管约30秒。样品将在毛细管的阴极端注入。为了避免不适当的样品注入,我们建议将真空泵放置在毛细管的阳极端
  4. 按照与步骤3相同的方式将0.5%HEC聚合物溶液填充到毛细管中(图2)。


    图2.将0.5%HEC聚合物溶液填充到毛细管中并清洁毛细管内壁的示意图

  5. 在Labview界面上设置CE参数:样品加载电压,样品加载时间,电场强度。当分析不同的样品时,这些参数应被优化以实现优选的分离
  6. 注入样品。如图3所示,将电源的阴极和毛细管的相应端浸入微量离心管中的样品溶液中。将电源的阳极和毛细管的相应端浸入含有0.5%HEC聚合物溶液的微离心管中。确保毛细管与电源良好连接。通过安装在计算机上的LabVIEW控制软件,设置在高压电源上。这样,高电压施加在毛细管上,样品被电动注入

    图3.注入样品的示意图

  7. 启动CE分离。取出样品微量离心管,并以??相同的方式放置含有0.5%HEC聚合物溶液的微量离心管。确保毛细管与电源连接良好。
  8. 在CE分离过程中,可以通过LabVIEW软件监测荧光信号。在第一次CE运行中,当观察到最后一个信号时,应保持CE过程持续另一个相同的持续时间,以确保没有其他信号出现。保存CE数据。继续数据分析。
  9. 在步骤3之后,清洁毛细管的内壁。清洁防止下一个CPE运行受到污染。如果不需要更多的CPE运行,它有助于保持毛细管长期储存

代表数据



图4.(a)dsRNA片段和(b)dsDNA片段在1.2%HEC(MW:1,300k)溶液中的电泳图。在100V/cm下进行毛细管电泳。毛细管的总长度和有效长度分别为9.0cm和6.0cm。将样品在100V/cm下加载2.0秒。使用0.5x TBE将dsDNA样品和dsRNA样品分别稀释到13ng /μl和20ng /μl。

笔记

  1. 变性对于dsDNA和dsRNA分子的电泳分离不是强制性的
  2. 您可以将dsDNA和dsRNA分析物保持在4°C进行短期储存,并将其保存在-20°C下长期储存。
  3. 分析物建议分解为0.5×TBE,以确保每次电动注射相同。
  4. 因为聚合物溶液是粘稠的并且需要很长时间才能得到均匀的分散,所以建议制备浓缩的聚合物溶液用于储存。在CPE实验之前,可以方便地将浓缩的聚合物溶液稀释至所需浓度。在这种情况下,我们演示了制备最终浓度为0.5x TBE和2x SYBR Green II的0.5%HEC聚合物溶液的方案。
  5. 各种MW中的HEC聚合物,例如90k-1,300k,可用于dsDNA和dsRNA分析。如果HEC聚合物的MW低,则建议高浓度用于dsDNA和dsRNA分离
  6. SYBR Green I也可用于dsDNA和dsRNA死亡
  7. 建议将毛细管保存在超纯水中进行储存,以保持涂层表面的性能
  8. 请确保高电压的纹波小于施加电压的0.1%,高电压的转换速率应大于20 V /μsec。这些参数通常写在产品规格中。
  9. 如果毛细管长,推??荐高泵压力或注射器,以便将粘稠的聚合物溶液填充到毛细管中。如果毛细管短,则在毛细管内形成气泡的情况下优选低压。在我们的实验室,共同的毛细管长度不超过20厘米。我们的真空泵的最终压力为26.6×10 3 Pa
  10. 超纯水系统应产生符合ASTM(美国测试材料学会)1.1型标准的水
  11. 我们相信,商业可用的CE系统与该协议兼容以适应实验过程。

食谱

  1. 储存1%HEC聚合物溶液,10ml
    1 g HEC粉末
    9 ml 0.5x TBE
    在室温下将溶液搅拌至少24小时,以获得均匀的聚合物溶液。将聚合物溶液保持在室温。
  2. 0.5x TBE 1,000 ml
    50 ml 10x TBE
    950毫升超纯水
    使用0.22μm滤膜过滤0.5x TBE溶液两次。
    保持室温。
  3. 100x SYBR Green
    1μl10,000x SYBR Green
    99μl超纯水
    保持在2-8°C至少一个月。

致谢

这项工作由Yamaguchi和Liu等人2015年修订和修订。
该项目得到了科学研究助理的部分支持[No. 25600049(Y.Y.),A15H038270(Y.Y)],JSPS,Japan。我们承认华东理工大学的部分资金支持(No. YK0142119)。

参考文献

  1. (a),(a),(b),(c),(c),(c),(d),(d)/p> 1229:对于毛细管电泳中的RNA分离,脉冲电场仍然有效吗? 1229: 274-279。
  2. Liu,C.,Yamaguchi,Y.,Zhu,X.,Li,Z.,Ni,Y.and Dou,X.(2015)。  通过毛细管电泳在羟乙基纤维素溶液中分析小干扰RNA。 36(14): 1651-1657。
  3. Sumitomo,K.,Sasaki,M。和Yamaguchi,Y。(2009)。  电泳 30(9):1538-1543。
  4. Todorov,TI,Yamaguchi,Y。和Morris,MD(2003)。  尿素对用于核酸电泳分离的聚合物缓冲溶液的影响。 Anal Chem 75(8):1837-1843。
  5. Yamaguchi,Y.,Todorov,TI,Morris,MD和Larson,RG(2004)。  在半纯和稀释的羟乙基纤维素溶液中单个DNA分子电泳迁移的分布。电泳 25(7-8):999-1006。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, C., Yamaguchi, Y. and Dou, X. (2016). Capillary Electrophoresis in Hydroxyethylcellulose Solutions for the Analysis of dsDNA, dsRNA, and siRNA. Bio-protocol 6(15): e1894. DOI: 10.21769/BioProtoc.1894.
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