搜索

3 users have reported that they have successfully carried out the experiment using this protocol.
Test MicroRNA Target with in vitro Cell Culture Luciferase Assay
采用体外细胞培养荧光素酶试验法测试MicroRNA靶标   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Nature Neuroscience
Aug 2012

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. This protocol is to be used to test the binding and activity of miRNA on putative UTR target sequences. It is based on the expression of Luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miRNA of interest or synthetic miRNA to test in an in vitro cell culture assay.

Keywords: MicroRNA activity (microRNA活性), 3'UTR (3’UTR), Luciferase assay (荧光素酶检测), In vitro (体外)

Materials and Reagents

  1. HEK293T cells (ATCC, catalog number: CRL-11268 TM)
  2. DMEM (High Glucose) (GutaMAXTM) (Life technologies, catalog number: 61965 )
  3. Fetal bovine serum (FBS) heat inactivated (Sigma-Aldrich, catalog number: F9665 )
  4. Penicillin-Streptomycin (Life technologies, catalog number: 15140 )
  5. pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, catalog number: E1330 )
  6. miScript miRNA Mimic (QIAGEN, catalog number varies depending on your interest miRNA)
  7. Lipofectamine®2000 Transfection Reagent (Life Technologies, catalog number: 11668 )
  8. Dual-Glo Luciferaseassay system (Promega Corporation, catalog number: E2920 )
  9. Cuiture medium (see Recipes)

Equipment

  1. Luminometer (Berthold Technologies)
  2. White 96-well plates (Greiner, catalog number: 655074 )

Procedure

  1. Clone the putative UTR sequence to test into the pmirGLO Dual-Luciferase miRNA Target Expression Vector.
  2. Plate 1 x 104 cells per well in 50 μl culture medium from a sub-confluent HEK293T cell suspension (cell density < 1.3 x 105 cells/cm2). Plate 3 wells for each condition to produce triplicates (conditions should include negative controls for miRNA and UTR sequence and positive control when existing such as known miRNA for the target or known target for the miRNA).
  3. Transfect cells after 5-16 h in culture. The following is transfection condition for 1 well:
    1. Dilute 300 ng of reporter plasmid (an example of vector is pmirGLO Dual-Luciferase miRNA Target Expression Vector) in combination with miRNA Mimic (such as miScript miRNAMimic) at a final concentration of 5 to 50 nM in 25 μl of pure DMEM.
    2. Dilute 0.5 μl Lipofectamine in 25 μl of pure DMEM.
    3. Incubate both diluted solutions 5 min at room temperature.
    4. Combine diluted nucleic acids with diluted Lipofectamine (50 μl final), mix gently and incubate complexes 20 min at room temperature.
    5. Add the transfection complexes (50 μl) to the well containing plated cells in 50 μl medium.
      Note: Usually cells are plated in the morning for a few hours and transfected in the afternoon. 20 nM is a usual working concentration for miRNA. Instead of mimic, plasmid encoding pre-miRNA could also be used (300 ng/well).
  4. Incubate transfected cells at 37 °C in a 5% CO2 incubator for 48 h without medium change.
  5. Measure luciferase using the Dual-Glo Luciferase assay system as recommended by the manufacturer:
    1. Prepare the stop solution, according to the number of wells, by diluting Dual-Glo® Stop &Glo®Substrate (1:100) in Dual-Glo® Stop &Glo® Buffer.
    2. Due to evaporation of the medium with time, the final volume per well is less than 100 μl after 48 hin culture. Measure the volume of the medium left in one well by pipetting and discard the appropriate amount of medium to reduce the volume to 50 μl from the well by pipetting out.
    3. Add 50 μl of Dual-Glo® Luciferase Reagent to each well (equal volumes). Avoid depositing Reagent solution along the walls of the well. Mix gently by tapping the plate. Because the plate is not shaken, when a drop of reagent is on the wall of the well, the volume of reagent in contact with the culture medium is reduced and this has an impact on the measurement of the luciferase.
    4. Incubate 10 min in the dark at room temperature without shaking.
    5. Measure firefly luciferase activity using the luminometer.
    6. Remove the plate from the luminometer and add 50 μl of Dual-Glo®Stop&Glo® Reagent (stop solution prepared in a.) per well. Mix gently.
    7. Incubate 10 min in the dark at room temperature.
    8. Measure Renilla luciferase activity using the luminometer.
      Note: Dual-Glo®Luciferase and Dual-Glo®Stop&Glo® Reagents are stable for 2 h.
  6. Calculate the ratio of luminescence from the experimental reporter (firefly) to luminescence from the control reporter (Renilla). Calculate the mean ratio for each triplicate and normalize this ratio to the ratio of control wells. Experimental conditions will include a miRNA negative control (which does not target the UTR), a reporter plasmid devoid of UTR or containing an irrelevant UTR sequence.

Recipes

  1. Culture medium
    DMEM, high glucose, GutaMAXTM
    10% FBS
    1% Penicillin-Streptomycin (optional, medium either with or without antibiotics will not affect transfection efficiency)

Acknowledgments

I thank Ute Bissels (Miltenyi Biotec) and Philipp Follert for providing the pmirGlo vector and for advice on luciferase assays. Funding was provided by the Agence National de la Recherche (ANR, FORDOPA), Fondation pour la Recherche Médicale (Label Equipe FRM), Fondation de France and the European Commission (Marie-Curie: ITN AXREGEN and IAPP DopaNew).

References

  1. de Chevigny, A., Core, N., Follert, P., Gaudin, M., Barbry, P., Beclin, C. and Cremer, H. (2012). miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons. Nat Neurosci 15(8): 1120-1126.

简介

微小RNA(miRNA)是长度为21-24个核苷酸的小非编码RNA,通过靶向mRNA的非翻译区(UTR)来调节基因表达。 该方案用于测试miRNA对推定的UTR靶序列的结合和活性。 它是基于荧光素酶作为报道基因的表达,其在含有目的前miRNA或合成miRNA的质粒存在下在UTR体外融合,以在体外细胞培养测定中测试。

关键字:microRNA活性, 3’UTR, 荧光素酶检测, 体外

材料和试剂

  1. HEK293T细胞(ATCC,目录号:CRL-11268 TM
  2. DMEM(高葡萄糖)(GutaMAX TM )(Life technologies,目录号:61965)
  3. 热灭活的胎牛血清(FBS)(Sigma-Aldrich,目录号:F9665)
  4. 青霉素 - 链霉素(Life technologies,目录号:15140)
  5. pmirGLO双荧光素酶miRNA靶表达载体(Promega Corporation,目录号:E1330)
  6. miScript miRNA Mimic(QIAGEN,目录号取决于您的兴趣miRNA)
  7. Lipofectamine 2000转染试剂(Life Technologies,目录号:11668)
  8. Dual-Glo Luciferase Assay系统(Promega 公司,目录号:E2920)
  9. 快餐介质(见配方)

设备

  1. 发光计(Berthold Technologies)
  2. 白色96孔板(Greiner,目录号:655074)

程序

  1. 克隆推定的UTR序列以测试进入pmirGLO双荧光素酶miRNA靶向表达载体
  2. 将来自亚汇合HEK293T细胞悬浮液(细胞密度<1.3×10 5细胞/cm 2)的50μl培养基中的每孔1×10 4个细胞/2 )。 板3孔,每种条件产生一式三份(条件应包括miRNA和UTR序列的阴性对照,以及存在时的阳性对照,例如已知的miRNA靶或miRNA的已知靶)。
  3. 在培养5-16小时后转染细胞。 以下是1孔的转染条件:
    1. 在25μl纯DMEM中将终浓度为5至50nM的300ng报道质粒(载体的实例是pmirGLO双荧光素酶miRNA靶表达载体)与miRNA模拟物(例如miScript miRNAMimic)一起稀释。
    2. 稀释0.5μlLipofectamine在25μl纯DMEM中
    3. 在室温下孵育两种稀释的溶液5分钟
    4. 将稀释的核酸与稀释的Lipofectamine(最终50μl)混合,轻轻混合,并在室温下孵育复合物20分钟。
    5. 将转染复合物(50μl)加入含有50μl培养基的平板细胞的孔中 注意:通常细胞在早上电镀几个小时,并在下午转染。 20 nM是miRNA的常用工作浓度。 代替模拟,也可以使用质粒编码pre-miRNA(300ng /孔)。
  4. 在37℃,5%CO 2培养箱中孵育转染细胞48小时,无培养基更换。
  5. 使用制造商推荐的Dual-Glo荧光素酶测定系统测量荧光素酶:
    1. 根据孔的数目,通过在Dual-Glo底物中稀释Dual-Glo Stop& Glo ?底物(1:100)来制备终止溶液。 ® Stop& Glo ® Buffer。
    2. 由于培养基随时间的蒸发,在48小时培养后每孔的终体积小于100μl。通过移液器测量一个孔中剩余的培养基的体积,并丢弃适量的培养基,通过移液器将体积从孔中减少至50μl。
    3. 向每个孔(等体积)中加入50μl的Dual-Glo 萤光素酶试剂。避免沿着孔壁沉积试剂溶液。通过轻拍板轻轻混合。因为板不摇动,当井壁上有一滴试剂时,与培养基接触的试剂的体积减少,这对荧光素酶的测量有影响。
    4. 在室温下在黑暗中孵育10分钟,不摇动。
    5. 使用光度计测量萤火虫萤光素酶活性。
    6. 从发光计中取出板,并加入50μl每孔的Dual-Glo</Stop& Glo</sup>试剂 轻轻混匀。
    7. 在室温下在黑暗中孵育10分钟。
    8. 使用光度计测量海肾荧光素酶活性
  6. 计算来自实验报告子(萤火虫)的发光与来自对照报告子(Renilla)的发光的比率。 计算每个三次重复的平均比例,并将该比率归一化为对照孔的比率。 实验条件将包括miRNA阴性对照(其不靶向UTR),缺乏UTR或含有不相关UTR序列的报告质粒。

食谱

  1. 培养基
    DMEM,高葡萄糖,GutaMAX TM
    10%FBS
    1%青霉素 - 链霉素(可选,含或不含抗生素的培养基不会影响转染效率)

致谢

我感谢Ute Bissels(Miltenyi Biotec)和Philipp Follert提供pmirGlo载体和荧光素酶测定的建议。 资金由法国国家广播公司(ANR,FORDOPA),法国基金会,法国基金会和欧洲委员会(Marie-Curie:ITN AXREGEN和IAPP DopaNew)提供。

参考文献

  1. de Chevigny,A.,Core,N.,Follert,P.,Gaudin,M.,Barbry,P.,Beclin,C.and Cremer,H。(2012)。 miR-7a调节Pax6控制前脑多巴胺能神经元的空间起源。 Nat Neurosci 15(8):1120-1126。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Coré, N. (2013). Test MicroRNA Target with in vitro Cell Culture Luciferase Assay. Bio-protocol 3(5): e420. DOI: 10.21769/BioProtoc.420.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。