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Polysome Preparation, RNA Isolation and Analysis
多核糖体的制备、RNA分离和分析   

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Abstract

During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

Materials and Reagents

  1. Cells (Neuroblastoma cell line SKN-SH)
  2. Protease inhibitors (Sigma-Aldrich, catalog number: P8340-5ML )
  3. DTT (Sigma-Aldrich, catalog number: 43815 )
  4. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
  5. Heparin (Sigma-Aldrich, catalog number: H3149 )
  6. Sucrose (Sigma-Aldrich, catalog number: S1888 )
  7. Phenol/chloroform/isoamyl alcohol (Life Technologies, Invitrogen™, catalog number: 15593-031 )
  8. Sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
  9. 1x PBS
  10. 1x Trypsin-EDTA, 0.05% Trypsin/0.53 mM EDTA (Cellgro, catalog number: 25-052-CV )
  11. RPMI-1640 (Hyclone, catalog number: SH30096.01 )
  12. Tris-Base (Thermo Fisher Scientific, catalog number: BP-152-1 )
  13. KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
  14. MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: T8787-250ML )
  16. DNAase/RNAase free Ethanol (Sigma-Aldrich, catalog number: E7023-500ML )
  17. DNAase/RNAase free water (BioExpress, catalog number: UPW-1000 )
  18. Ultracentrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
  19. Polysome extraction buffer (PEB) (see Recipes)
  20. Sucrose solutions (see Recipes)

Equipment

  1. BR-188 Density gradient fractionation system (Brandel)
  2. Beckman optima L-70 ultracentrifuge (Beckman)
  3. 7500 Real-time PCR system (Applied Biosystems)
  4. Boekel scientific orbitron rotator I, 115 V (Boekel Scientific)

Procedure

  1. Prepare neuroblastoma cell line SKN-SH cells 2.5-5 x 107/group, normally 2 to 3 150 cm2 flasks with 50 ml of RPMI-1640 medium.
  2. Before harvest, cells are incubated in complete RPMI-1640 medium (RPMI-1640 medium with FBS, Pen/Strep and Glutamine) containing a final concentration of 100 μg/ml CHX for 10 min at 37 °C.
  3. Discard medium andrinse cells twice with cold 10 ml 1x PBS containing 100 μg/ml CHX.
  4. Harvest cells, briefly add 7 ml of trypsin into each flask, incubate at 37 °C for 3 min, then collect cells with 10 ml PBS containing 100 μg/ml CHX (this step is not needed for suspension cells).
  5. After centrifugation, resuspend cells in 1 ml of 1x PBS, then transfer them into 1.7 ml micro centrifuge tube.
  6. Lyse cells with 1 ml PEB Buffer containing 1% Triton-X100, vortex 15 sec and keep on ice for 30 min.
  7. Centrifuge at 14,000 rpm for 30 min at 4 °C and collect supernatants that are ready to be loaded on sucrose gradients of 10%-50% consistency.
  8. Make sucrose solutions according to the recipe step.
  9. Make 10 ml gradient by adding 2.0 ml of each solution (50% on bottom, 10% on top) and load into ultracentrifuge tube.
  10. Add 1 ml of cell extract to the top of each gradient. Cover each tube with parafilm, put it in a SW41 rotor andspin samples at 38,000 rpm for 120 min at 4 °C.
  11. Collect 13 fractions from the top to the bottom of the tube using BR-188 density gradient fractionation system.
  12. Machine setting options: fraction collection time: 1 min and 15 sec/fraction; pump speed, 0.75 ml/min; chart speed, 60 cm/h).
  13. Keep all fractions at -80 °C, it’s good for extraction of RNA in 1 month.
  14. For RNA extraction, thaw all samples on ice.
  15. Pick 0.5 ml of each sample into a new 2 ml-microcentrifuge tube, keep on ice.
  16. Mix the water saturation Phenol/chloroform/isoamyl alcohol (25:24:1) completely.
  17. Add 500 μl of the mixed Phenol/chloroform/isoamyl alcohol into each tube, when pipetting keep shaking with hand to avoid water separating out.
  18. Vortex for 30 sec and then keep rotating at 4 °C for 5 min.
  19. Centrifuge at 14,000 rpm for 10 min at 4 °C, at the same time prepare new 2 ml-microcentrifuge tube for each sample, add 3 M Sodium Acetate (pH 5.2) 50 μl/tube.
  20. Very carefully pipet out all the aqueous phase and put it into the prepared fresh 2 ml-microcentrifuge tube.
  21. Then add 1.5 ml (3 volumes) of 100% ethanol, vortex 30 sec.
  22. Keep the samples at -80 °C for 3 h.
  23. Centrifuge at 14,000 rpm for 30 min at 4 °C.
  24. Wash pellet with cold 70% RNase free ethanol by rotating for 5 min.
  25. Centrifuge pellet at 14,000 rpm for 5 min at 4 °C.
  26. Discard supernatants and remain the pellet in the tubes.
  27. Put tubes on ice to let all the liquid evaporated.
  28. Add 30-50 μl of DNase/RNase free H2O and subjected to quantitated RT-PCR.


Figure 1. MDM2 regulates MYCN translation. A. Representative polysomal profiles from control vector- and MDM2/166A-transfected SK-N-SH cells. The 254-nm traces obtained during collection of fractions are shown. B. Relative distributions of MYCN and GAPDH mRNA in SK-N-SH cells transfected either with vehicle or MDM2/166A.

Recipes

  1. Polysome extraction buffer (PEB)
    (Triton-X100 Free)
    20 mM Tris-HCl (pH 7.5)
    50 mM KCl
    10 mM MgCl2
    1 mM DTT
    100 μg/ml CHX
    200μg/ml Heparin
  2. Preparation of 50%, 40%, 30%, 20%, 10% sucrose solutions as following
    Make 50% Sucrose solution (25 g sucrose + PEB without Triton-X100 to 50 ml)
    Dilute 50% sucrose solution into the solutions of 40%, 30%, 20% and 10% in PEB buffer

Acknowledgments

This work was supported by the National Institutes of Health (R01 CA123490 and R01CA143107 to MZ) and CURE (MZ and LG).

References

  1. Gu, L., Zhang, H., He, J., Li, J., Huang, M. and Zhou, M. (2012). MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells. Oncogene 31(11): 1342-1353.

简介

在mRNA翻译期间,40S和60S核糖体亚基结合靶mRNA形成80S复合物(单体)。 这种核糖体在翻译延长过程中沿着mRNA移动以促进tRNA阅读密码子,其中翻译被激活并且许多单体可以同时结合相同的mRNA,其形成多核糖体。 多聚体可以通过蔗糖密度梯度离心进行大小分级。 多核糖体中更特异的mRNA意味着mRNA的更有活性的翻译状态。

材料和试剂

  1. 细胞(神经母细胞瘤细胞系SKN-SH)
  2. 蛋白酶抑制剂(Sigma-Aldrich,目录号:P8340-5ML)
  3. DTT(Sigma-Aldrich,目录号:43815)
  4. 环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)
  5. 肝素(Sigma-Aldrich,目录号:H3149)
  6. 蔗糖(Sigma-Aldrich,目录号:S1888)
  7. 苯酚/氯仿/异戊醇(Life Technologies,Invitrogen TM,目录号:15593-031)
  8. 乙酸钠(Thermo Fisher Scientific,目录号:S209-500)
  9. 1x PBS
  10. 1x胰蛋白酶-EDTA,0.05%胰蛋白酶/0.53mM EDTA(Cellgro,目录号:25-052-CV)
  11. RPMI-1640(Hyclone,目录号:SH30096.01)
  12. Tris-Base(Thermo Fisher Scientific,目录号:BP-152-1)
  13. KCl(Thermo Fisher Scientific,目录号:BP-366-500)
  14. MgCl 2(Sigma-Aldrich,目录号:M-2393)
  15. Triton X-100(Sigma-Aldrich,目录号:T8787-250ML)
  16. 无DNA酶/RNA酶的乙醇(Sigma-Aldrich,目录号:E7023-500ML)
  17. DNA酶/RNA酶游离水(BioExpress,目录号:UPW-1000)
  18. 超速离心管(14×89mm)(Beckman Coulter,目录号:344059)
  19. 聚合体提取缓冲液(PEB)(参见配方)
  20. 蔗糖溶液(见配方)

设备

  1. BR-188密度梯度分馏系统(Brandel)
  2. Beckman optima L-70超速离心机(Beckman)
  3. 7500实时PCR系统(Applied Biosystems)
  4. Boekel科学轨道旋转器I,115V(Boekel Scientific)

程序

  1. 用50ml RPMI-1640培养基制备成神经细胞瘤细胞系SKN-SH细胞2.5-5×10 7个/组,通常为2〜3150cm 2烧瓶。 />
  2. 在收获前,将细胞在含有终浓度为100μg/ml CHX的完全RPMI-1640培养基(含有FBS,Pen/Strep和谷氨酰胺的RPMI-1640培养基)中在37℃下孵育10分钟。
  3. 弃去培养基,并用含有100μg/ml CHX的冷10ml 10ml 1x PBS洗涤细胞两次
  4. 收获细胞,简单地加入7毫升胰蛋白酶到每个烧瓶中,在37℃孵育3分钟,然后收集细胞与10毫升PBS含有100微克/毫升CHX(此步骤不需要悬浮细胞)。
  5. 离心后,将细胞重悬在1ml的1×PBS中,然后转移到1.7ml微量离心管中
  6. 裂解细胞与1毫升PEB缓冲液含1%Triton-X100,涡旋15秒,冰上保持30分钟。
  7. 在4℃以14,000rpm离心30分钟,收集准备装载在10%-50%浓度的蔗糖梯度上的上清液。
  8. 根据配方步骤制备蔗糖溶液。
  9. 通过加入2.0ml每种溶液(50%在底部,10%在顶部)使10ml梯度,并加载到超速离心管中。
  10. 加入1ml细胞提取物到每个梯度的顶部。 用石蜡膜覆盖每个管,将其置于SW41转子中,并在4℃下以38,000rpm旋转120分钟。
  11. 使用BR-188密度梯度分馏系统从管的顶部到底部收集13个级分。
  12. 机器设置选项:馏分收集时间:1分钟和15秒/分数; 泵速度,0.75ml/min; 图表速度,60厘米/小时)。
  13. 将所有级分保存在-80°C,适合在1个月内提取RNA
  14. 对于RNA提取,将所有样品在冰上融化
  15. 将每份样品0.5 ml放入新的2 ml离心管中,保存在冰上
  16. 完全混合水饱和度苯酚/氯仿/异戊醇(25:24:1)
  17. 在每个试管中加入500μl混合的苯酚/氯仿/异戊醇,当移液时用手保持摇动,避免水分离。
  18. 涡旋30秒,然后在4℃保持旋转5分钟。
  19. 在4℃下以14,000rpm离心10分钟,同时为每个样品制备新的2ml微量离心管,加入50μl/管的3M乙酸钠(pH5.2)。
  20. 非常小心地吸取所有的水相,并将其放入准备好的新鲜2毫升微量离心管中
  21. 然后加入1.5ml(3体积)的100%乙醇,涡旋30秒
  22. 保持样品在-80℃下3小时
  23. 在4℃下以14,000rpm离心30分钟。
  24. 通过旋转5分钟用冷的70%无RNA酶的乙醇洗涤沉淀
  25. 在4℃下以14,000rpm离心沉淀5分钟
  26. 弃去上清液,将沉淀物留在试管中。
  27. 将管放在冰上,让所有的液体蒸发
  28. 加入30-50μl不含DNase/RNase的H 2 O并进行定量RT-PCR。


图1. MDM2调节MYCN翻译。 A。 来自对照载体和MDM2/166A转染的SK-N-SH细胞的代表性多核糖体谱。 显示在收集级分期间获得的254nm迹线。 B.用载体或MDM2/166A转染的SK-N-SH细胞中MYCN和GAPDH mRNA的相对分布。

食谱

  1. 多聚体提取缓冲液(PEB)
    (Triton-X100 Free)
    20mM Tris-HCl(pH7.5) 50 mM KCl
    10mM MgCl 2/
    1 mM DTT
    100μg/ml CHX
    200μg/ml肝素
  2. 如下制备50%,40%,30%,20%,10%蔗糖溶液 制备50%蔗糖溶液(25g蔗糖+ PEB,不含Triton-X100至50ml) 将50%蔗糖溶液稀释到PEB缓冲液中的40%,30%,20%和10%的溶液中

致谢

这项工作由国家卫生研究院(R01 CA123490和R01CA143107到MZ)和CURE(MZ和LG)支持。

参考文献

  1. Gu,L.,Zhang,H.,He,J.,Li,J.,Huang,M.and Zhou,M.(2012)。 MDM2调节人神经母细胞瘤中MYCN mRNA的稳定和翻译 cell Oncogene 31(11):1342-1353。
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  • 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: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, H. and Zhou, M. (2012). Polysome Preparation, RNA Isolation and Analysis. Bio-protocol 2(21): e286. DOI: 10.21769/BioProtoc.286.
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sara ali
manchester university
Hi
I work with yeast and I have got polysomes fractions from my colleque and I want to converst them to cDNA using RT_PCR kit
Can you please help me wich primer should I use??
11/19/2013 7:30:38 AM Reply
Hailong Zhang
Emory University

Hi, Sara, we used the kit "iScript? Reverse Transcription Supermix for RT-qPCR" from Bio-rad for reverse transcription, please see the link for more detailed information:
https://www.bio-rad.com/en-us/sku/170-8840-iscript-reverse-transcription-supermix-for-rt-qpcr

Good luck

11/24/2013 9:07:55 PM


Agata Giallongo
IBIM-CNR
My question/comment concerns the use of Heparin in the Polysome Extraction Buffer (PEB):
heparin is not mentioned in the material and methods section of the full-length paper (see reference)where Rnasin 500U/ml) seems to have been routinely used.

It is known that heparin may interfere with downstream applications like RT and qPCR..... is it advisable not to use it at all?

AG
9/5/2013 8:52:49 AM Reply
Hailong Zhang
Emory University

Hi,Agata, it's a good question. but you don't worry about the downstream applications of RT,RT-PCR,qPCR,because RNA will be precipitated during RNA extraction(step:"21"),and all solutions will be washed away totally(step 24-27). RNA will be very pure and without any heparin any longer.
We need heparin during cell lysis, because the DNA binding sites on RNA polymerase can be occupied by heparin, preventing the polymerase from binding to promoter DNA.

9/10/2013 2:21:25 PM