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Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.

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Polysome Profiling Analysis

Molecular Biology > RNA > mRNA translation
Authors: Masahiro Morita
Masahiro MoritaAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
Bio-protocol author page: a708
Tommy Alain
Tommy AlainAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
Bio-protocol author page: a709
Ivan Topisirovic
Ivan TopisirovicAffiliation: Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada
For correspondence: ivan.topisirovic@mcgill.ca
Bio-protocol author page: a707
 and Nahum Sonenberg
Nahum SonenbergAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
For correspondence: nahum.sonenberg@mcgill.ca
Bio-protocol author page: a710
Vol 3, Iss 14, 7/20/2013, 10003 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.833

[Abstract] Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.

Keywords: Translation, Polysome, MRNA, Ribosome, Genome-wide analysis

Materials and Reagents

  1. Sucrose
  2. 1 M HEPES-KOH (pH 7.6)
  3. 2 M KCl
  4. 1 M MgCl2
  5. PBS
  6. 1 M Tris-HCl (pH 7.5)
  7. 10 mg/ml cycloheximide (Sigma-Aldrich, catalog number: C7698) in MilliQ water
    Note: Cycloheximide inhibits protein synthesis by blocking translation elongation. This molecule interferes with the translocation of tRNAs with the mRNA and the ribosome, resulting in fixed ribosomes on mRNAs.
  8. Protease inhibitor cocktail (EDTA-free) (F. Hoffmann-La Roche, catalog number: 04 693 159 001)
  9. RNase inhibitor (RNasin 40 units/μl) (Promega Corporation, catalog number: N2511)
  10. 10% Triton X-100
  11. 10% Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750)
  12. RNaseZap (Life Technologies, Ambion®, catalog number: AM9780)
  13. Trizol (Life Technologies, InvitrogenTM)
  14. Oligo dT primer and Super Script III (Life Technologies, InvitrogenTM, catalog number: 18418012 and 18080093)
  15. 10x sucrose gradient buffer (see Recipes)
  16. 10-50% sucrose solutions (see Recipes)
  17. Hypotonic buffer (see Recipes)

Equipment

  1. Ultra centrifuge and rotor (Beckman, model: Optima L-80; SW40Ti rotor)
  2. UV detector and fraction collector (Teledyne ISCO)
  3. NanoDrop (Thermo Fisher Scientific)
  4. 0.22 µm filter

Software

  1. TracerDAQ software (MicroDAQ)

Procedure

I.   Preparation of sucrose gradients

  1. Prepare 50 ml of 60% (w/v) sucrose solution in MilliQ water and 10 ml of 10x sucrose gradient buffer. Filter solutions with 0.22 μm filter.
  2. Prepare 10-50% sucrose solutions using 60% sucrose. Prepare gradients at least one day before cell lysis to allow gradient to diffuse overnight at 4 °C.
  3. Carefully add 1.2 ml of each sucrose solution (starting with the 50% sucrose solution) to Beckman tubes (12 ml total volume).To improve efficacy at this step, the gradients can be flash frozen in liquid nitrogen following each addition of sucrose solution. This helps in generating equally poured gradients and prevents too much diffusion.
  4. Cover tube with parafilm and incubate at 4 °C overnight. Alternatively, sucrose gradients can be stored at -80 °C indefinitely.


II.  Isolation of polysomes

  1. Prepare cells at least one day before lysing. Confluency is very important. About 80-90% confluency gets the maximum polysomes. 1-5 15-cm dishes are needed to see polysome profile.
  2. Prior to lysing the cells, treat cells with 100 μg/ml cycloheximide at a final concentration for 5 min.
  3. Wash cells twice with 10 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide.
  4. Scrape cells with 5 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide, collect them in 15 ml tube and rinse with 5 ml of ice-cold 1x PBS containing 100 μg/ml cycloheximide.
  5. Centrifuge cells at 1,200 rpm (300 x g) for 5 min at 4 °C.
  6. Aspirate supernatant, resuspend cells in 425 μl of hypotonic buffer and transfer all to a new pre-chilled 1.5 ml tube.
  7. Then add:
    5 μl of 10 mg/ml cycloheximide
    1 μl of 1 M DTT
    100 units of RNasin
  8. Vortex for 5 sec.
  9. Then add:
    25 μl of 10% Triton X-100
    25 μl of 10% Sodium Deoxycholate
  10. Vortex for 5 sec.
  11. Centrifuge at 15,000 rpm (21,000 x g, a maximal speed on a bench top centrifuge in 1.5 ml eppendorf tube) for 5 min at 4 °C.
  12. Transfer supernatant (about 500 μl) to a new pre-chilled 1.5 ml tube. Measure OD260nm for each sample using NanoDrop.


    Figure 1. Polysome profiling from MEFs treated with DMSO or Ink1341 (250 nM) for 8 h from Dowling et al. (2010). 40 S, 60 S and 80 S denote the corresponding ribosomal subunits and monosome, respectively.

  13. Load the same OD amount of lysate onto each gradient (10-30 OD260nm is loaded). Keep 10% of lysates as an input.
  14. Weight and balance each gradient.
  15. Centrifuge at 35,000 rpm for 2 h at 4 °C using SW40Ti rotor in a Beckman Coulter (Optima L-80 ultra centrifuge). No brake of brake at 1 (maximum) for deceleration. Acceleration does not matter much.
  16. While the samples are centrifuging, clean fraction collector with warm MilliQ water containing a bit of RNase ZAP (a few sprays).
  17. Carefully remove tubes from the rotor and place them at 4 °C until they are ready for running.
  18. Switch on computer, pump, UV detector and fraction collector. Set pump at 1.5 ml/min and the fraction collector by time. Place 2 ml tubes on fraction collector.
  19. Open TracerDAQ program.
  20. Run chasing solution (60% (w/v) sucrose with bromophenol blue) through the system until it reaches the needle. Make sure to see at least one drop coming out of the needle such that no bubbles are introduced into the gradient.
  21. Screw each tube onto the ISCO UV detector and then pierce the tube with the needle.
  22. Begin running the chasing solution through the gradient. Run solution with the pump at 1.5 ml/min. Click on acquire data button and press run on the fraction collector.
  23. Place fractions on dry ice or liquid nitrogen.
  24. Add 750 μl of Trizol for RNA isolation.


III. RNA isolation and RT-qPCRMQ

  1. Add 150 μl of chloroform to each fraction.
  2. Isolate RNA according to manufacturer’s Trizol protocol.
  3. Measure the RNA concentration of each fraction or input.
  4. Prepare the following solutions in 500 μl tubes or PCR tubes.
    0.5 μg of RNA                     x μl
    Oligo (dT) primer                1 μl
    dNTP (10 mM each)           1 μl
    Water x μl/total                   13 μl
  5. 65 °C for 5 min.
  6. On ice for at least 1 min.
  7. Add the following solutions (7 μl premix) on ice. Make premix before adding.
    5x buffer                    4 μl
    1 M DTT                    1 μl
    RNasin 40 units/μl     1 μl
    RTase                       1 μl
  8. Mix gently and spindown on ice.
  9. 50 °C for 60 min.
  10. 70 °C for 15 min.
  11. Keep at 4 °C until next step.
  12. Add 180 μl of MQ.
  13. qPCR according to manufacturer’s protocol.
    2x SYBR                  10 μl
    5’ Primer (10 μM)     0.4 μl
    3’ Primer (10 μM)     0.4 μl
    RT sample               5 μl
    MQ 4.2 μl/Total        20 μl

Recipes

  1. 10x sucrose gradient buffer (50 ml)
    200 mM HEPES (pH 7.6)
    1 M KCl
    50 mM MgCl2
    100 μg/ml cycloheximide
    1x protease inhibitor cocktail (EDTA-free)
    100 units/ml RNase inhibitor
  2. 10-50% sucrose solutions
    Final (%)
    60% stock (ml)
    water (ml)
    10x sucrose gradient buffer (ml)
    50
    8.3
    0.7
    1
    45
    7.5 1.5
    1
    40
    6.7
    2.3
    1
    35
    5.8
    3.2
    1

    30
    5.0
    4.0
    1

    25
    4.2
    4.8
    1

    20
    3.3
    5.7
    1

    15
    2.5
    6.5
    1

    10
    1.7
    7.3
    1

  3. Hypotonic buffer
    5 mM Tris-HCl (pH 7.5)
    2.5 mM MgCl2
    1.5 mM KCl
    1x protease inhibitor cocktail (EDTA-free)

References

  1. Alain, T., Morita, M., Fonseca, B. D., Yanagiya, A., Siddiqui, N., Bhat, M., Zammit, D., Marcus, V., Metrakos, P., Voyer, L. A., Gandin, V., Liu, Y., Topisirovic, I. and Sonenberg, N. (2012). eIF4E/4E-BP ratio predicts the efficacy of mTOR targeted therapies. Cancer Res 72(24): 6468-6476. 
  2. Dowling, R. J., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B. D., Petroulakis, E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y., Kozma, S. C., Thomas, G. and Sonenberg, N. (2010). mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 328(5982): 1172-1176.
  3. Larsson, O., Morita, M., Topisirovic, I., Alain, T., Blouin, M. J., Pollak, M. and Sonenberg, N. (2012). Distinct perturbation of the translatome by the antidiabetic drug metformin. Proc Natl Acad Sci U S A 109(23): 8977-8982. 


How to cite: Morita, M., Alain, T., Topisirovic, I. and Sonenberg, N. (2013). Polysome Profiling Analysis. Bio-protocol 3(14): e833. DOI: 10.21769/BioProtoc.833; Full Text



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12/23/2015 10:03:21 PM  

Yingying Lin
Xiamen University

Dear all,
I am a doctoral student of Xiamen University in China. Recently, our lab bought a BioComp gradient machine. And I have done several times of polysome profile of MCF-7 cell line in order to set up the technique. However, the result didn't comes out good. Here I present a graph of the latest polysome profile I did. I use the protocol you provide. I used about 370 ug of RNA to load to the 5~50% sucrose gradient and centrifuge at 36,000 rpm for 2 hr. The polysome peaks didn't drop down to the baseline at the end, and after 80S the wave didn't go down as well. What do you think the problem is? Do you have some advice for me to improve the technique? Thank you so much!


Best regards,

Ying

 
12/23/2015 10:18:52 PM  

Ivan Topisirovic (Author)
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital,McGill University

Looks like you are overloading the gradient and/or that there be something wrong with your gradients. What we typically load is about 10OD at 254nm. Note that we use Biocomp gradient master but we fractionate using ISCO collector and not Biocomp (so we can't advise on Biocomp), but this shouldn't be an issue. This should help, as here you have the detailed protocol (with things that may go wrong) and whole video on how to do it.

http://www.ncbi.nlm.nih.gov/pubmed/24893926

Hope this helps, and if not I'll transfer you to Morita as I haven't done one of these in a while.

12/23/2015 10:39:21 PM  

Yingying Lin
Xiamen University

Dear Ivan,
Thank you for your fast reply. Actually, I have seen the jove video you mentioned. And I done the polysome profile according to it as well, because I see your protocol is similar as that. You mean I overload the RNA to the gradient, I don't understand what is 10OD at 254 mean, I have measure the RNA with nanodrop, the OD 260 is 18.198, should I dilute the sample to adjust the OD260 to be 10, and how much volume of the sample should I load to the gradient? We use the sw41i rotor. On the other hand, there was something wrong with my gradient, what do you think is wrong?
Many thanks!

Best regards,

Ying

12/23/2015 10:56:19 PM  

Ivan Topisirovic (Author)
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital,McGill University

I have never seen a profile like yours so it's hard to say what you're doing wrong.

Yes I would load 10 OD from 260, and if you make a gradient using Biocomp gradient master you just have to make sure that it's continuous 5-50% (you can read the manual if you're confused re: zonal vs isopycnic centrifugation and it's crucial that you use rate zonal and not ispycnic caps). These are two things that I would try, and see whether they'd help.

If you followed the JOVE video and protocol it tells you exactly how much to load:

"Transfer ultracentrifuge tubes containing sucrose gradients in pre-chilled rotor buckets. Remove 500 µl from the top of sucrose gradients. Adjust lysates so that they contain the same OD (10-20 OD at 260 nm) in 500 µl of lysis buffer (described in step 2.5) and load them onto each sucrose gradient. Note: It is important to immediately load lysates on the gradients, as this will critically improve the quality of polysome preparations."

So it seems to me that you could follow it a bit more carefully, and according to my experience if you can't find this information in the protocol that's written as clearly as JOVE or the one here, then there may be a problem of how careful you are when you do the experiment. So try to be more careful.

12/23/2015 11:11:16 PM  

Yingying Lin
Xiamen University

Dear Ivan,
Thank you very much! I am so embarrased to say that I saw the sentence and it mentioned 500 ul of the sample, for the engineer who set up our machine told me to add to full of the centrifuge tube, so for several times I only load 300 ul of the sample. So I see, maybe I should just remove 500 ul of the gradient and add 500 ul of the sample. Thank you again for your advice!


Best wishes,

Ying

12/25/2015 2:50:01 AM  

Yingying Lin
Xiamen University

Dear Ivan,
Today, I did another polysome profile according to the protocol of jove paper, I used 10OD 260(500 uL) RNA sample to 5~50% sucrose gradient and centrifuge at 36,000 rpm for 2 hr, but the result still not good. Accidently, I cut the impurity peak, but the wave after 80S didn't go down, I can't figure out what's wrong with it, can you help me and give me some advice? Many thanks!


Best regards,

Ying

 
12/25/2015 6:56:46 AM  

Ivan Topisirovic (Author)
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital,McGill University

If you followed the protocol, then then the only thing I can think of is that this could be due to the way you make the gradient or how the collector is set up, so I'd suggest you contact Biocomp. I can't help you any further as I have no idea what's going on. Never seen a profile like this. Good luck with the experiments and sorry I couldn't help.

12/25/2015 7:02:09 AM  

Yingying Lin
Xiamen University

Nevermind,you've helped me a lot! Many thanks!

3/22/2016 5:05:17 AM  

laura GA
isciii

Dear Ivan,
Thank you for the protocol. I have tried it with MCF7 cells (4 of 15-cm dishes). After of lysis, the OD 260 is around 2. What is the problem?. Also, I tried it with 293T cells (10 of 10-cm dishes) and the OD 260 was 1.5 . I believe the problem doesn´t the cell number because I have a lot of cells. When I run these RNA samples in a sucrose gradient I get a good profile but with low OD 254. Then, the problem is the celular lysis?
Best

Reply

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1/23/2015 5:26:39 AM  

Carmine Onofrillo
Bologna University

Dear all,
i'm a post doc of bologna university. Recently we bought a teledyne isco gradient system, provided with UA6 UV detector, optical unit, and a fraction collector for polysome profile analysis. In your protocols you indicate the tracerdaq as the software used for computetional data recording. I'm trying to connect UA6 detector to a personal computer to have digitalized data. I have some problems setting the tracerdaq software and the coupled hardware device, the minilab 1008. In particular i don't know how to connect output recorders of the UA6 detector to the minilab 1008, and how to set the voltage range of tracerdaq. Can you help me in some way?
Many thanks and regards.
Carmine Onofrillo.

1/26/2015 11:54:47 AM  

Ivan Topisirovic (Author)
Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital,McGill University

Dear Carmine,

In our lab, USB-1208L is used to connect the UA6 detector to the laboratory computer. To connect the UA6 detector to USB digital I/O, this protocol may be of use (http://www.isco.com/pcfiles/PartPDF/SL000004/UP000ZUZ.pdf). Moreover, TracerDAQ software is included in USB-1208L with the protocol about the connection (http://www.microdaq.com/measurement_computing/usb_data_acquisition/multifunction/usb-1208/usb-1208ls-daq.php). If you need any further information, please do not hesitate to contact us.

All the best,

Masahiro Morita

1/27/2015 1:34:48 AM  

Carmine Onofrillo
Bologna University

Dear dott. Morita,
thanks for your fast reply. Actually i'm not sure about the connection of the cable from recorder output of UA6 he UA6 manual, detector to my device . Our device (as yours) can operate in single ended mode ( only +/- 10V) or in differential mode (+/- 10, 5, 2.5 ,2, 1.25, 1V), in single ended i have to use only 2 cable (POSITIVE and GROUND), in differential i have to connect 3 cables ( HI, LOW and GROUND). Do you know what settings i have to use? In The tracerdaq software and in instacal it is possible to check the type of connection (single ended or differential), in the settings menu. I hope you can help me.
Best regards,
Carmine Onofrillo.

1/28/2015 7:14:29 PM  

Masahiro Morita (Author)
Department of Biochemistry and Goodman Cancer Research Centre,McGill University

Dear Carmine,

I would like to send the photos of UA6 detector and USB I/O adaptor to you. Would it be possible to let me know your email address?

All the best,

Masahiro Morita

Reply

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