搜索

Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction
从麝香百合(百合)花药分离小孢子并提取RNA   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

This protocol has been designed in order to facilitate the isolation and extraction of total RNA from microspores collected from lily anther sacs. This protocol allows the extraction of high amounts of high quality RNA, as observed in agarose gels.

Keywords: Microspore separation(微孢子分离), RNA extraction(RNA提取), Lily plant(百合植物)

Material and Reagents

  1. Young anthers (at the microspore stage of lily buds normally from 30 to 65 mm of bud length)
  2. Lithium chloride (LiCl) (Merck KGaA, catalog number: 105679 )
  3. Tris-base (United States Biological, catalog number: 75825 )
  4. Ethylenediaminetetraacetic acid (EDTA) (United States Biological, catalog number: 15699 )
  5. Sodium dodecyl sulfate (SDS) (AppliChem, catalog number: A2263 )
  6. Isoamylalcohol (J.T. Baker®, catalog number: JT9038-1 )
  7. Phenol (pH 4.0) (United States Biological, catalog number: 77510 )
  8. Chloroform (Merck KGaA, catalog number: 102445 )
  9. Deionized distilled water (ddH2O)
  10. Diethylpyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 )
  11. Sodium acetate (NaOAc) (Sigma-Aldrich, catalog number: S2889 )
  12. Isopropanol (Merck KGaA, catalog number: 107022 )
  13. 75% ethanol (analytical grade)
  14. Liquid nitrogen
  15. 0.1% DEPC ddH2O (RNase-free water) (see Recipes)
  16. Extraction buffer (see Recipes)
  17. 3 M sodium acetate (see Recipes)
  18. 10 mM sodium acetate buffer (see Recipes)
  19. 4 M LiCl (see Recipes)
  20. 75% ethanol (see Recipes)

Equipment

  1. Scalpel
  2. Tweezers (stainless, length: 11.5 cm; tip: 0.1 mm)
  3. Ceramic mortar and pestle
  4. 1.5 ml Eppendorf
  5. Centrifuge (Eppendorf, model: 5424 )
  6. Fume hood
  7. Speed vacuum (Savant Systems LLC, model: SC110 )

Procedure

  1. Microspore collection
    1. Approximately 25 anthers of young lily buds (3.5 cm) are transversely sliced close to the edge of an anther with a scalpel.
    2. The locular fluid (microspore) is collected in a 1.5 ml Eppendorf tube containing 200 μl of 10 mM sodium acetate buffer (pH 5.2) from the cutting site using a tweezer that gently squeezes out the fluid by moving along the surface of the anther to the cutting side.

      Video 1. Separation of Microspore

    3. Centrifuge at 7,000 rpm (4,600 x g) for 5 min.
    4. Decant the supernatant and the pellet (microspore) in the tube is saved for RNA extraction.

  2. RNA isolation
    1. Immerse the Eppendorf tube that contains the microspore pellet in liquid nitrogen.
    2. Transfer the frozen microspores (0.2 g) into mortar and pestle in liquid nitrogen and grind the sample into powder.
    3. Work in the fume hood. The tissue powder is added by 1 ml of a mix of hot (~80 °C) extraction buffer/phenol (1:1) and continually ground in a mortar in liquid nitrogen.
    4. After melting, the solution of sample mixture is transferred to a 1.5 ml Eppendorf tube.
    5. Add 0.2 ml of chloroform/isoamylalcohol (24:1, v/v) into an Eppendorf tube, close the lid, and hand-shake the tube vigorously for 30 sec.
    6. Centrifuge the sample at 12,000 rpm (13,500 x g) for 5 min at 4 °C.
    7. Transfer the solution of the upper phase to a new Eppendorf tube and add an equal volume of 4 M LiCl.
    8. Precipitate RNA at -80 °C for 2 h or -20 °C overnight (approximately 16 h) (Note 1).
    9. Centrifuge at 12,000 rpm for 10 min at 4 °C.
    10. Decant the supernatant.
    11. The RNA pellet is dissolved in 0.4 ml of RNase-free water by pipetting up and down for several times.
    12. Add 1/10 volume (40 μl) of 3 M sodium acetate (pH 5.2) and 1 volume (0.4 ml) of isopropanol.
    13. Precipitate RNA at -80 °C for 2 h or -20 °C overnight (approximately 16 h) (Note 1).
    14. Centrifuge at 12,000 rpm for 5 min at 4 °C.
    15. Decant the supernatant.
    16. Wash pellet with 0.6~1 ml of 75% ethanol by pipetting up and down for several times.
    17. Centrifuge at 12,000 rpm for 5 min at 4 °C.
    18. Decant the supernatant.
    19. Shortly dry the RNA pellet by speed vacuum.
    20. Dissolve RNA with an appropriate amount of RNase-free water (usually 20 μl or less) and incubate at 55-60 °C for 10 min.
    21. The RNA sample is stored at -80 °C.

Representative data



Figure 1. Total RNA (20 μg) was isolated from microspores isolated from lily flower buds with different lengths. 1. 34-36 mm buds; 2. 44-46 mm buds, both are at the microspore stage and 3. 60-65 mm buds at the transition between microspore and mature pollen. Total RNA was denatured, fractionated on a formaldehyde-agarose gel and stained with ethidium bromide.

Recipes

  1. 0.1% DEPC ddH2O (RNase-free water)
    1 ml of DEPC is added into 999 ml of ddH2O, mixed at 37 °C for 1 h and autoclaved.
  2. Extraction buffer                                            100 ml          final conc.
    4 M LiCl
    5 ml
    200 mM
    1 M Tris-HCl (pH 8.0)
    20 ml
    200 mM
    0.5M EDTA
    4 ml
    5 mM
    10% SDS
    20 ml
    2%
    DEPC
    ddH2O
    51 ml
  3. 3 M sodium acetate (pH 5.2)
    24.61 g of sodium acetate (MW = 82.03) is added into 100 ml of ddH2O, pH is adjusted to 5.2, and autoclaved.
  4. 10 mM sodium acetate buffer (pH 5.2)
    82.03 mg of sodium acetate (MW= 82.03) is added into 100 ml of ddH2O, pH is adjusted to 5.2, and autoclaved.
  5. 4 M LiCl
    16.96 g of LiCl (MW= 42.39) is added into 100 ml of ddH2O and autoclaved.
  6. 75% ethanol
    789.5 ml of 95% ethanol is added into 210.5 ml ddH2O.

Notes

  1. Precipitation of RNA at -20 °C overnight (approximately 16 h) may lead to a better yield of total RNA. However, if yield is not a concern, most RNAs can be precipitated at -20 °C for 2 h.

Acknowledgments

The protocol is an adaption from the protocol of Verwoerd et al. (1989). We greatly appreciate their original contribution.

References

  1. Liu, M. C., Yang, C. S., Yeh, F. L., Wei, C. H., Jane, W. N., Chung, M. C. and Wang, C. S. (2014). A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development. J Exp Bot 65(8): 2023-2037.
  2. Verwoerd, T. C., Dekker, B. M. and Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17(6): 2362.

简介

该协议已被设计以便于从从百合花囊收集的小孢子中分离和提取总RNA。 这个协议允许大量的高质量RNA,如在琼脂糖凝胶中观察到的提取。

关键字:微孢子分离, RNA提取, 百合植物

材料和试剂

  1. 年轻花药(在小孢子阶段的百合芽通常从30至65毫米的芽长)
  2. 氯化锂(LiCl)(Merck KGaA,目录号:105679)
  3. Tris-base(United States Biological,目录号:75825)
  4. 乙二胺四乙酸(EDTA)(United States Biological,目录号:15699)
  5. 十二烷基硫酸钠(SDS)(AppliChem,目录号:A2263)
  6. 异戊醇(J.T.Baker ,目录号:JT9038-1)
  7. 苯酚(pH4.0)(United States Biological,目录号:77510)
  8. 氯仿(Merck KGaA,目录号:102445)
  9. 去离子蒸馏水(ddH 2 O)
  10. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758)
  11. 乙酸钠(NaOAc)(Sigma-Aldrich,目录号:S2889)
  12. 异丙醇(Merck KGaA,目录号:107022)
  13. 75%乙醇(分析纯)
  14. 液氮
  15. 0.1%DEPC ddH 2 O(无RNase水)(参见配方)
  16. 提取缓冲液(参见配方)
  17. 3 M乙酸钠(见配方)
  18. 10 mM乙酸钠缓冲液(见配方)
  19. 4 M LiCl(参见配方)
  20. 75%乙醇(见配方)

设备

  1. Scalpel
  2. 镊子(不锈钢,长度:11.5cm;尖端:0.1mm)
  3. 陶瓷砂浆和杵
  4. 1.5 ml Eppendorf
  5. 离心机(Eppendorf,型号:5424)
  6. 通风橱
  7. 高速真空(Savant Systems LLC,型号:SC110)

程序

  1. 微孢子收集
    1. 将约25个年轻百合芽的花药(3.5cm)用手术刀在花药的边缘附近横向切片。
    2. 将该局部流体(小孢子)收集在1.5ml Eppendorf中                 管中含有200μl10mM乙酸钠缓冲液(pH 5.2)                 切割位点使用镊子轻轻地挤出流体                 沿着花药的表面移动到切割侧

      视频1.分离微孢子
                                                                                                                                                                                                                                                               <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                                  

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >
    3. 以7,000rpm(4,600×g)离心5分钟
    4. 倾析上清液,保存管中的沉淀(小孢子)用于RNA提取

  2. RNA分离
    1. 将含有小孢子丸的Eppendorf管浸泡在液氮中
    2. 将冷冻的小孢子(0.2g)转移到研钵中,在液氮中研磨,并将样品研磨成粉末
    3. 在通风橱工作。 通过1ml混合物加入组织粉末                  的热(〜80℃)萃取缓冲液/苯酚(1:1)并连续研磨                  在液氮中的研钵
    4. 熔化后,将样品混合物的溶液转移到1.5ml Eppendorf管中
    5. 加入0.2ml氯仿/异戊醇(24:1,v/v)                  Eppendorf管,关闭盖子,并手动摇动管30天                  秒。
    6. 在4℃下,以12,000rpm(13,500xg)离心样品5分钟。
    7. 将上层溶液转移到新的Eppendorf管中,加入等体积的4M LiCl
    8. 在-80℃下沉淀RNA 2小时或-20℃过夜(约16小时)(注1)。
    9. 在4℃下以12,000rpm离心10分钟。
    10. 倾析上清液。
    11. 通过上下吹打数次,将RNA沉淀溶解在0.4ml无RNA酶的水中
    12. 加入1/10体积(40μl)的3M乙酸钠(pH5.2)和1体积(0.4ml)异丙醇。
    13. 在-80℃下沉淀RNA 2小时或-20℃过夜(约16小时)(注1)。
    14. 在4℃下以12,000rpm离心5分钟。
    15. 倾析上清液。
    16. 用0.6〜1ml的75%乙醇通过上下吹打数次来洗涤沉淀物
    17. 在4℃下以12,000rpm离心5分钟。
    18. 倾析上清液。
    19. 通过速度真空快速干燥RNA沉淀。
    20. 用适量的无RNA酶的水(通常20μl或更少)溶解RNA,并在55-60℃孵育10分钟。
    21. 将RNA样品储存在-80℃。

代表数据



图1.从具有不同长度的百合花蕾分离的小孢子中分离总RNA(20μg)。 1.34-36mm芽; 2. 44-46 mm芽,两者均处于小孢子阶段,3.60-65mm芽处于小孢子和成熟花粉之间的转变处。 使总RNA变性,在甲醛 - 琼脂糖凝胶上分级并用溴化乙锭染色。

食谱

  1. 0.1%DEPC ddH 2 O(无RNase水)
    将1ml DEPC加入到999ml ddH 2 O中,在37℃下混合1小时并高压灭菌。
  2. 撷取缓冲区                             ;                  100 ml           最终浓度。
    4 M LiCl
    5 ml
    200 mM
    1 M Tris-HCl(pH 8.0)
    20ml
    200 mM
    0.5M EDTA
    4 ml
    5 mM
    10%SDS
    20ml
    2%
    DEPC
    ddH2O
    51 ml
  3. 3 M乙酸钠(pH 5.2)
    将24.61g乙酸钠(MW = 82.03)加入100ml ddH 2 O中,将pH调节至5.2,并高压灭菌。
  4. 10mM乙酸钠缓冲液(pH5.2) 将82.03mg乙酸钠(MW = 82.03)加入100ml ddH 2 O中,将pH调节至5.2,并高压灭菌。
  5. 4 M LiCl
    将16.96g LiCl(MW = 42.39)加入到100ml ddH 2 O中并高压灭菌。
  6. 75%乙醇 将789.5ml的95%乙醇加入到210.5ml ddH 2 O中

笔记

  1. 在-20℃下沉淀RNA过夜(约16小时)可以导致更好的总RNA产量。 然而,如果不关心产量,大多数RNA可以在-20℃下沉淀2小时。

致谢

该协议是来自Verwoerd等人(1989)的协议的改编。 我们非常感谢他们的贡献。

参考文献

  1. Liu,M.C.,Yang,C.S.,Yeh,F.L.,Wei,C.H.,Jane,W.N.,Chung,M.C.and Wang,C.S。(2014)。 一种新颖的百合花药特异性基因编码在花药发育过程中与外壁形成相关的粘附素样蛋白。/a> J Exp Bot 65(8):2023-2037。
  2. Verwoerd,T.C.,Dekker,B.M.and Hoekema,A。(1989)。 快速分离植物RNA的小规模程序核酸 Acids Res 17(6):2362.
  • 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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, M., Chen, Y. and Wang, C. (2015). Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction. Bio-protocol 5(4): e1400. DOI: 10.21769/BioProtoc.1400.
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google 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。