Polysome Analysis

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Mar 2016



Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA.

Keywords: Translation (翻译), Ribosome fractionation (核糖体分离), Polysome (多聚核糖体), mRNA (mRNA), Sucrose density gradient (蔗糖密度梯度), Ultracentrifugation (超速离心)


The cellular mRNAs are distributed into an actively translating and a non-translating pool at any point of time and can dynamically redistribute between these pools in response to various stimuli. The actively translating mRNAs have a higher number of ribosomes associated with them and the number of ribosome associated with an mRNA is a measure of the translation state of the mRNA. Therefore on fractionating the ribosomes from a cell, actively translating mRNAs will be found in the polysomal fraction whereas non-translating/poorly-translating mRNAs will be either found in the free mRNA fraction or associated with 40S ribosomal subunits. Polysome analysis is therefore a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes (Ray et al., 2009; Poria et al., 2016). Association of individual mRNAs with translating/non-translating fractions can be detected by RT-PCR, whereas the entire translation or non-translating pool of mRNAs can be identified by RNA sequencing or microarray analysis.

Materials and Reagents

  1. Ultracentrifuge tube for SW41Ti (Seton Scientific, catalog number: 7030 )
  2. Permanent ink marker
  3. 10 ml syringe with wide gauge luer lock cannula (Vita Needles, gauge 14)
  4. 10 cm dish (Eppendorf, catalog number: 0030702115 )
  5. 1.5 ml tube (RNase and DNase free) (Corning, Axygen®, catalog number: MCT-150-C or equivalent)
  6. MCF7 human breast carcinoma cell line (ATCC, catalog number: HTB22 )
  7. Cycloheximide (CHX) (AMRESCO, catalog number: 94271 )
  8. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  9. DEPC treated water (AMRESCO, catalog number: E174 )
  10. Citrate saturated phenol (pH 4.5) (Sigma-Aldrich, catalog number: P4682 )
  11. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  12. 75% ethanol
  13. Nuclease free water
  14. Oligo dT primer
  15. dNTP mix
  16. 5x RT buffer
  17. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  18. RNase inhibitor (Thermo Fisher Scientific, catalog number: EO0381 )
  19. cDNA synthesis reagents (Thermo Fisher Scientific, InvitrogenTM, catalog number: 28025013 )
  20. 10x PCR buffer (with 15 mM MgCl2)
  21. 10 µM forward primer for gene of interest/control gene
  22. 10 µM reverse primer for gene of interest/control gene
  23. PCR reagents (New England Biolabs, catalog number: M0273L )
  24. Sucrose (RNase and DNase free) (AMRESCO, catalog number: 0335 )
  25. Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
  26. Magnesium chloride (MgCl2) (AMRESCO, catalog number: 0288 )
  27. HEPES (pH 7.4) (Thermo Fisher Scientific, Affymetrix, catalog number: 16926 )
  28. IGEPAL CA-360 detergent (Sigma-Aldrich, catalog number: I3021 )
  29. Protease inhibitor cocktail (AMRESCO, catalog number: M250 )
  30. 10% sucrose solution (for 8 ml) (see Recipes)
  31. 50% sucrose solution (for 8 ml) (see Recipes)
  32. Polysome lysis buffer (see Recipes)


  1. Gradient Station or any equivalent gradient fractionators (Biocomp, catalog number: 153-002 )
  2. Tube holder
  3. Ultra-centrifuge (Beckman Coulter, model: Optima L-70 or equivalent)
  4. Rotor SW41Ti (Beckman Coulter, catalog number: 331362 )
  5. 200 µl pipette
  6. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  7. Fraction collector (Gilson, model: FC203B )
  8. UV monitor (Bio-Rad Laboratories, model: EM1-EconoTM )
  9. Balance
  10. Vortex


  1. Gradient Master software (included with the Gradient Station)


  1. Gradient preparation
    1. Prepare 8 ml of 10% and 50% sucrose solution each in 1x sucrose gradient buffer for a single gradient (see Recipes, Table 1).
    2. Degas the solution under vacuum for 5 min.
    3. Mark ultracentrifuge tubes at 50% of tube height with permanent ink marker using the marker block provided with the Gradient Station.
    4. Carefully add 10% sucrose solution upto the 50% mark of the tube with a wide gauge cannula.
    5. Aspirate the 50% sucrose solution into a syringe and insert the cannula directly to the bottom of the tube through the 10% sucrose solution then slowly layer the heavier solution to the bottom of the tube without disturbing the 10% sucrose solution. The heavier sucrose solution will slowly push the 10% solution up, and the 50% sucrose solution should reach up to the 50% marking of the ultracentrifuge tube.
    6. Cover the tube with rubber cap and place the tube in the tube holder on the rotor of the gradient station.
    7. Run the following pre-set program in the Gradient Master software
      Program name: Short Sucrose 10-50% (w/v) Fast
      Program specification: 10-50%; Step: S1/1; Time: 1 min 45 sec; Angle: 81.5; rpm: 25 (Video 1).
    8. Carefully take out the tubes and store vertically at 4 °C for 2 h without disturbance.

      Video 1. Gradient preparation

  2. Cell lysate preparation for polysome profiling
    1. Treat 70-80% confluent cells in a 10 cm dish with 100 µg/ml cycloheximide (CHX) by directly adding it to the culture media at 15 to 30 min prior to harvesting.
    2. Wash the cells twice with PBS containing 100 µg/ml CHX, and harvest them by scraping into 10 ml ice cold PBS containing 100 µg/ml CHX. Centrifuge at 600 x g for 5 min at 4 °C and discard the supernatant carefully.
    3. Lyse the cell pellet by adding 200 µl of polysome lysis buffer (see Recipes). Mix gently with 200 µl pipette for a number of times and incubate on ice for 15 min.
    4. Centrifuge at 10,000 x g for 20 min at 4 °C.
    5. Transfer the supernatant to a pre-chilled 1.5 ml tube.
    6. Measure OD at 254 nm using a spectrophotometer.
    7. Overlay 25-50 OD equivalent of lysate gently on top of the gradient in the ultracentrifuge tube without disturbing the gradient. Leave around 2 mm of space in the gradient tube on top of the lysate layer (Figure 1).
    8. Weigh each gradient tube in a balance and adjust the weight of each tube containing the gradient to be exactly equal by adding polysome lysis buffer to the overlaid layer without disturbing the gradient.

      Figure 1. 10%-50% sucrose gradient showing 2 mm space on top of gradient

  3. Ultracentrifugation and fraction collection
    1. Pre-cool the rotor assembly and the tube buckets at 4 °C.
    2. Load the tubes carefully into the buckets without disturbing the gradients. Load the buckets on the rotor assembly ensuring equal weights in opposing positions. Perform the ultracentrifugation at 111,000 x g (Avg RCF) for 4 h at 4 °C at maximum vacuum.
    3. During the last hour of the ultracentrifugation switch on the gradient profiler, UV detector, gradient profiling software and wash the fractionator tubing with running DEPC treated water for several times and dry by running air.
    4. Carefully take out the tubes, one at a time, from the buckets and load into the tube holder of the gradient fractionator, and place at the proper position under the collector piston.
    5. Run water through the tubing and press auto zero on the UV monitor.
    6. Set up the tube length, gradient length (75 cm for SETON open top polyclear tube), fraction number (12 to 16) in the set up window.
    7. Run the fractionation and collect fractions (Figure 2) in 1.5 ml tubes and transfer to ice immediately.

      Figure 2. Ribosomal fractions from MCF7 cells were analysed by 10-50% sucrose density gradient fractionation. A. Ribosomal RNA content, measured at 254 nm, is plotted against fraction numbers (top panel). B. RNA isolated from selected fractions was analyzed by semiquantitative RT-PCR using PDCD4 and GAPDH primers (bottom panel).

  4. RNA isolation and RT-PCR
    1. Add 300 µl phenol (citrate saturated, pH 4.5) and 300 µl chloroform to the tubes containing the gradient fractions.
    2. Vortex for 30-60 sec.
    3. Centrifuge at 16,000 x g for 15 min at 4 °C.
    4. Carefully transfer the supernatants to new tubes.
    5. Add 10 µg glycogen (RNase free) and equal volume of isopropanol and mix by inverting the tubes several times. Incubate at room temperature for 30 min.
    6. Centrifuge at 16,000 x g for 30 min at 4 °C.
    7. Wash the RNA pellet with 1 ml ice cold 75% ethanol.
    8. Air dry the RNA pellet and dissolve into 10-20 µl of nuclease free water.
    9. Measure the absorbance at 260 nm in a spectrophotometer to determine RNA concentration.
    10. cDNA synthesis: Setup the following reaction for cDNA synthesis for each RNA.
      8 µl
      Oligo dT primer
      1 µl
      10 mM dNTP mix
      1 µl
      65 °C for 5 min. Keep on ice for 1 min.
      5x RT buffer
      4 µl
      100 mM DTT 
      2 µl
      RNase inhibitor
      0.5 µl
      M-MLV RT (200 U/µl)
      1 µl
      Nuclease free water
      2.5 µl
      Incubate at 37 °C for 1 h. Then at 70 °C for 15 min.
      Store at 4 °C.
    11. PCR
      10x PCR buffer (with 15 mM MgCl2)
      2 µl
      10 µM forward primer for gene of interest/control gene
      1 µl
      10 µM reverse primer for gene of interest/control gene
      1 µl
      Taq DNA polymerase (1 U/µl)
      1 µl
      2 µl
      Nuclease free water
      13 µl
      Run thermal cycling for 30-35 cycles (Denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for 30 sec) and run the products on agarose gel (Figure 2).


  1. 10% sucrose and 50% sucrose solution (for 8 ml) (Table 1)

    Table 1. Recipe for sucrose density gradient preparation

    10% sucrose solution (for 8 ml) 
    50% sucrose solution (for 8 ml)
    0.8 g
    4.0 g
    2 M KCl
    400 µl
    400 µl
    1 M MgCl2
    40 µl
    40 µl
    100 mM DTT
    160 µl
    160 µl
    1 M HEPES pH 7.4
    160 µl
    160 µl
    100 mg/ml Cycloheximide
    8 µl
    8 µl
    DEPC water
    Upto 8 ml
    Upto 8 ml

  2. Polysome lysis buffer
    100 mM KCl
    5 mM MgCl2
    20 mM HEPES (pH 7.4)
    0.5% NP-40
    100 μg/ml CHX
    2 mM DTT
    40 U/ml RNase inhibitor
    1x protease inhibitor cocktail


We thank members of our laboratory for trying out and standardizing this protocol. Research which led to the development of these protocols was funded by a Wellcome Trust-DBT India Alliance Intermediate fellowship (WT500139/Z/09/Z) to PSR and a CSIR, India Senior Research Fellowship to DKP. This protocol is modified from the protocol described in Merrick and Hensold (2001).


  1. Merrick, W. C. and Hensold, J. O. (2001). Analysis of eukaryotic translation in purified and semipurified systems. Curr Protocols Cell Biolo 11-9.
  2. Poria, D. K., Guha, A., Nandi, I. and Ray, P. S. (2016). RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4. Oncogene 35(13): 1703-1715.
  3. Ray, P. S., Jia, J., Yao, P., Majumder, M., Hatzoglou, M. and Fox, P. L. (2009). A stress-responsive RNA switch regulates VEGFA expression. Nature 457(7231): 915-919.



背景 细胞mRNA在任何时间点分布到主动翻译和非翻译池中,并可响应于各种刺激而在这些池之间动态地重新分布。主动翻译的mRNA具有较高数量的与它们相关的核糖体,与mRNA相关的核糖体数量是mRNA的翻译状态的量度。因此,从细胞中分离核糖体时,会在多核糖体组分中发现主要转录的mRNA,而在游离mRNA部分或与40S核糖体亚基相关的非翻译/不良翻译mRNAs。因此,多聚赖氨酸分析是根据其与多核糖体的关联将mRNA从细胞分离为主动翻译和非翻译部分的方法(Ray et al。,2009; Poria等人, >。,2016)。可以通过RT-PCR检测单个mRNA与翻译/非翻译级分的关联,而通过RNA测序或微阵列分析可以鉴定mRNA的整个翻译或非翻译池。

关键字:翻译, 核糖体分离, 多聚核糖体, mRNA, 蔗糖密度梯度, 超速离心


  1. SW41Ti超级离心管(Seton Scientific,目录号:7030)
  2. 永久性墨迹
  3. 10毫升注射器,带宽量规鲁尔锁套管(Vita针,量规14)
  4. 10厘米盘(Eppendorf,目录号:0030702115)
  5. 1.5ml管(RNase和DNase free)(Corning,Axygen ,目录号:MCT-150-C或同等物)
  6. MCF7人乳腺癌细胞系(ATCC,目录号:HTB22)
  7. 环己酰亚胺(CHX)(AMRESCO,目录号:94271)
  8. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  9. DEPC处理水(AMRESCO,目录号:E174)
  10. 柠檬酸饱和酚(pH 4.5)(Sigma-Aldrich,目录号:P4682)
  11. 氯仿(Sigma-Aldrich,目录号:C2432)
  12. 75%乙醇
  13. 核酸酶免费水
  14. Oligo dT引物
  15. dNTP mix
  16. 5x RT缓冲区
  17. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  18. RNase抑制剂(Thermo Fisher Scientific,目录号:EO0381)
  19. cDNA合成试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:28025013)
  20. 10×PCR缓冲液(含15mM MgCl 2)
  21. 10μM正向引物用于感兴趣的基因/对照基因
  22. 10μM反向引物用于感兴趣的基因/对照基因
  23. PCR试剂(New England Biolabs,目录号:M0273L)
  24. 蔗糖(RNase和DNA酶免费)(AMRESCO,目录号:0335)
  25. 氯化钾(KCl)(AMRESCO,目录号:0395)
  26. 氯化镁(MgCl 2)(AMRESCO,目录号:0288)
  27. HEPES(pH 7.4)(Thermo Fisher Scientific,Affymetrix,目录号:16926)
  28. IGEPAL CA-360洗涤剂(Sigma-Aldrich,目录号:I3021)
  29. 蛋白酶抑制剂鸡尾酒(AMRESCO,目录号:M250)
  30. 10%蔗糖溶液(8 ml)(见食谱)
  31. 50%蔗糖溶液(8 ml)(见配方)
  32. Polysome裂解缓冲液(参见食谱)


  1. 梯度站或任何等效梯度分馏器(Biocomp,目录号:153-002)
  2. 管夹
  3. 超离心机(Beckman Coulter,型号:Optima L-70或相当的)
  4. 转子SW41Ti(Beckman Coulter,目录号:331362)
  5. 200微升移液管
  6. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  7. 馏分收集器(Gilson,型号:FC203B)
  8. UV监视器(Bio-Rad Laboratories,型号:EM1-Econo TM
  9. 平衡
  10. 涡流


  1. Gradient Master软件(随附梯度台)


  1. 渐变准备
    1. 准备8毫升10%和50%的蔗糖溶液,每个在1x蔗糖梯度缓冲液中单一梯度(见配方,表1)。
    2. 将溶液在真空下脱气5分钟
    3. 使用渐变站提供的标记块,使用永久性墨迹标记50%管高度的超速离心管。
    4. 用宽量规插管小心地加入10%蔗糖溶液至管的50%标记。
    5. 将50%蔗糖溶液吸入注射器,并通过10%蔗糖溶液将插管直接插入管的底部,然后缓慢地将较重的溶液层叠到管的底部,而不会干扰10%的蔗糖溶液。较重的蔗糖溶液将缓慢推入10%溶液,50%蔗糖溶液应达到超离心管50%的标记。
    6. 用橡胶盖盖住管子,将管子放在梯度台转子上的管座上
    7. 在Gradient Master软件
      中运行以下预设程序 程序名称:短蔗糖10-50%(w/v)快速
      程序规格:10-50%;步骤:S1/1;时间:1分45秒;角度:81.5; rpm:25(视频1)。
    8. 仔细取出管,并在4°C垂直存放2 h,不受干扰。

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  2. 多核糖体分析的细胞裂解物制备
    1. 在收获之前15-30分钟直接将培养基添加到培养基中,用100μg/ml的放线菌酮(CHX)将10%培养皿中的70-80%汇合细胞处理。
    2. 用含有100μg/ml CHX的PBS洗涤细胞两次,并通过刮入含有100μg/ml CHX的10ml冰冷的PBS中收获。在4℃下以600×x离心5分钟,小心地弃去上清液。
    3. 通过加入200μl多聚体裂解缓冲液(参见食谱)来溶解细胞沉淀。用200微升移液管轻轻混合多次,并在冰上孵育15分钟。
    4. 在4℃下以10,000×g离心20分钟。
    5. 将上清液转移到预先冷却的1.5ml管中
    6. 使用分光光度计测量254nm处的OD。
    7. 在超离心管中的梯度顶部轻轻地覆盖25-50 OD的裂解物,而不会影响梯度。在裂解层顶部的梯度管中留下2毫米的空间(图1)
    8. 称量每个梯度管平衡,并通过向重叠的层添加多聚体裂解缓冲液,将含有梯度的每个管的重量调整为完全相等,而不影响梯度。

      图1. 10%-50%蔗糖梯度,显示梯度上方的2mm空间

  3. 超速离心和馏分收集
    1. 转杯组件和管桶在4°C预冷。
    2. 将管仔细装入桶中,不会影响梯度。将铲斗装在转子组件上,确保相对位置的重量相等。在最大真空度下,在4℃下以111,000 x g(Avg RCF)进行超速离心4小时。
    3. 在梯度轮廓仪超紫外探测器的最后一小时内,使用紫外检测器,梯度分析软件,并用运行中的DEPC处理过的水清洗分馏器多次,并通过运行空气进行干燥。
    4. 仔细取出管子,一次一桶地从桶中取出,并装入梯度分馏器的管架中,并放置在收集器活塞下方的适当位置。
    5. 通过管道运行水,并在UV监视器上按自动调零。
    6. 设置管长,梯度长度(SETON打开顶部多毛管为75厘米),设置窗口中的分数(12至16)。
    7. 运行分馏并收集1.5 ml管中的馏分(图2),并立即转移至冰上

      图2.通过10-50%蔗糖密度梯度分级分析来自MCF7细胞的核糖体级分。A.在254nm测量的核糖体RNA含量相对于分数编号(上图)。 B.使用PDCD4和/或GAPDH引物(底部图),通过半定量RT-PCR分析从选定级分分离的RNA。

  4. RNA分离和RT-PCR
    1. 向含有梯度级分的管中加入300μl苯酚(饱和柠檬酸盐,pH4.5)和300μl氯仿
    2. 漩涡30-60秒
    3. 在4℃下以16,000xg离心15分钟。
    4. 小心地将上清液转移到新管上
    5. 加入10μg糖原(不含RNA酶)和等体积的异丙醇,并将管反转多次混合。在室温下孵育30分钟
    6. 在4℃下以16,000 x g离心30分钟。
    7. 用1ml冰冷的75%乙醇洗涤RNA沉淀
    8. 空气干燥RNA沉淀,溶解于10-20μl无核酸酶的水中
    9. 在分光光度计中测量260nm处的吸光度,以确定RNA浓度。
    10. cDNA合成:为每个RNA设定以下cDNA合成反应。
      Oligo dT引物
      10 mM dNTP mix
      1 μl
      5x RT缓冲区
      100 mM DTT 
      M-MLV RT(200U /μl)
      在37℃孵育1小时。然后在70°C 15分钟 在4°C储存。
    11. PCR
      10×PCR缓冲液(含15mM MgCl 2) 2μl
      10μM正向引物用于感兴趣的基因/对照基因 1μl
      10μM反向引物用于感兴趣的基因/对照基因 1μl
      DNA聚合酶(1U /μl)


  1. 10%蔗糖和50%蔗糖溶液(8ml)(表1)


    10%蔗糖溶液(8 ml) 
    50%蔗糖溶液(8 ml)
    0.8 g
    2 M KCl
    1 M MgCl 2
    100 mM DTT
    1 M HEPES pH 7.4
    高达8 ml
    高达8 ml

  2. 多聚裂解酶缓冲液
    100 mM KCl
    5mM MgCl 2
    20 mM HEPES(pH 7.4)
    100μg/ml CHX
    2 mM DTT
    40 U/ml RNase抑制剂


我们感谢我们实验室的成员尝试和规范这个协议。导致这些协议发展的研究由惠康信托 - 印度联盟中级联盟(WT500139/Z/09/Z)向PSR和CSIR,印度DKP高级研究奖学金资助。该协议从Merrick和Hensold(2001)中描述的协议修改。


  1. Merrick,WC和Hensold,JO(2001)。< a class ="ke-insertfile"href ="http://onlinelibrary.wiley.com/doi/10.1002/0471143030.cb1109s08/abstract;jsessionid=396749528FD7B4FE7711BD9BD64F7ED8.f02t03 ?userIsAuthenticated = false&deniedAccessCustomisedMessage ="target ="_ blank">纯化和半纯化系统中的真核翻译分析 Curr Protocols Cell Biolo 11-9。
  2. Poria,DK,Guha,A.,Nandi,I. and Ray,PS(2016)。  RNA结合蛋白HuR螯合微小RNA-21以防止炎症性肿瘤抑制基因程序性细胞死亡的翻译抑制4. 致癌基因 35(13):1703 -1715。
  3. Ray,PS,Jia,J.,Yao,P.,Majumder,M.,Hatzoglou,M.and Fox,PL(2009)。  应激反应RNA开关调节VEGFA表达。自然 457(7231):915-919。
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引用:Poria, D. K. and Ray, P. S. (2017). Polysome Analysis. Bio-protocol 7(6): e2192. DOI: 10.21769/BioProtoc.2192.



Runlai Hang
University of California, Riverside
Dear Author, I would like to know whether the DNase treatment is needed for the RNA before further RT reaction?
12/19/2017 6:43:53 PM Reply
Dipak Poria
National Cancer Institute

Hello Runlai, DNase digestion is not necessary for the RNA before RT reaction as cytoplasmic fraction is used for the polysome analysis in this protocol. Cell lysate preparation step in this protocol removes nucleus from the cytoplasmic fraction. Best, Dipak

12/20/2017 2:40:25 PM