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
Equipment
Software
Procedure
I. Preparation of sucrose gradients
II. Isolation of polysomes
III. RNA isolation and RT-qPCRMQ
Recipes
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
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/24893926Hope this helps, and if not I'll transfer you to Morita as I haven't done one of these in a while.
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
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.
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
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
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.
Nevermind,you've helped me a lot! Many thanks!
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
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
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.
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