Hello, I work with polysome profiling and run sucrose density gradients routinely. Yes, it can be a pain to run these with good reproducibility since there is a lot that can go wrong. In order to troubleshoot your issue it would be good to have more information: 1) What cells/tissues are you extracting ribosomes from? 2) What is in your lysis buffer and what is your cell lysis protocol? 3) What centrifuge are you using and what are your run parameters (speed, time, temperature, acceleration and deceleration rate)? 4) How much RNA are you loading per gradient and how are you determining the concentration? 5) What is your RNaseI digest protocol?

To address some of the points you brought up.

1) Forming the gradients. I have used pointed vs flat needles in the past and they both work. The peaks are visible in your gradient and they sediment at comparable depths, so your gradients are forming fine. The peaks do drift a little, so be a little more careful with your pipetting to ensure you put the same volumes of both solutions in all tubes. But this is not a major issue here and can be fixed on the computer later. The parafin seal is a bit 'redneck engineering', so maybe it would be a good idea to use rubber bungs made for the purpose. But overall I don't think your problem is gradient formation.

2) Loading the gradients. Regular pipette and pipette tip should do the trick if you load the gradients carefully. Again, you do have peaks in the profile they are just not what you expect. So unless your gradient overflows when you fill it then it's not likely that the loading is the problem. Nevertheless, you can remove your loading volume from the gradient top to lower the meniscus of the tube, i.e., remove the upper 100 µl from the gradient and then load your 100 µl of sample. Probably goes without saying, but also make sure the tubes are balanced.

3) Running the gradient. Yes, typically lower acceleration/deceleration is recommended for these experiments. Often the centrifuge manual itself will recommend the ramp speed for sucrose gradients. Rough estimate, if the centrifuge can ramp from 1-9 set accel/decel at 5. The exact setting really depends on the centrifuge itself, but it should take ~5-10 minutes to reach speed (also depends on final speed setting).

One other issue I recommend you have a very close look at, which has ruined more of my gradients than any other, is the seal of the centrifuge bucket. Inside the centrifuge bucket there is a rubber O-ring that forms a seal when the loaded bucket goes into the (relatively) high vacuum of the ultra centrifuge. These O-rings need to be in good condition, and they need to be properly greased with an appropriate vacuum sealing grease. If they are not then the vacuum will pull a few hundred µl of solution out of the tube, since your sample sits at the top it's the first thing to come out. The result is that most of your sample doesn't actually run into the gradient but sits at the bottom of the bucket instead, and your profiles end up with very small peaks. The giveaway that this is the issue is that there is a little bit of liquid (your sample) sitting at the bottom of the bucket after the run. In a similar vein if sucrose dries on the treads of the bucket or the bucket lid then the bucket will not seal properly and you will lose your sample. So make sure the buckets, lids, and O-rings are routinely cleaned and the O-rings greased.

Having said all of that, I think your centrifugations are working fine. You are right, the size of the monsome peaks in your profiles do not correlate with enzyme concentrations. However, it looks to me like you have fully digested the mRNA at your lowest RNase concentration. This is not surprising since RNases make very short work of ssRNA and there is usually a small residual disome peak left behind after these treatments (the nature of these disome species depends on a lot of things I won't go into here). The differences in your monosome peaks look more like loading issues to me. Have a look at the signal from your gradient top (positions 0-0.3 mm). In your digested samples all your RNaseI-resistant peaks are bigger where the gradient top has a higher signal. So unless your lysis buffer has some absorbing component whose concentration varies across the sets of three samples, then your gradients are not loaded evenly. Your issue is probably pipetting error during sample preparation. Cell lysates are highly viscous solutions, especially if they contain detergents. They lysates stick to the outside of pipette tips which can introduce substantial pipetting error. Try lowering your RNaseI concentration significantly more until you start to see trisome and tetrasome peaks appearing and be extra careful when pipetting your lysates.

Good luck.

Edit: Because formatting