To get a better idea what may have happend, it would be good if you could describe your sample, how much you used per mg/cell number, how you treated your sample before...

In general as soon as you add the reagent (Phenol / Guanidinisothiocyanat mixture), your RNA should be safe. So there are two time points that are really crucial for your RNA: The sample preparation itself and the elution/handling afterwards.

What I write now is just generally since I don't have any more information about your sample. The most common problem students had with isolating RNA from tissue is that they didn't mince the pieces small enough before adding the reagent (we are using TriFast Gold mostly), so the enzymes are not inactivated fast enough. This is not a real problem with tissues like brain or lung but highly relevant for intestines and stomach, just for some examples. 

With RNA isolation from cells there were other problems. First the cells were already in solution and too much fluid was added to the reagent. It is quite nice to see because the pH dependent colour change of the reagent. For whole fresh and untreated whole blood samples times was always the biggest issue. As soon as the blood starts clogging, you have RNA degradation. For blood samples from mice (we didn't want to treat the blood) we simply had the reagent added to the tube before drawing blood and then just released the blood sample in the reagent. 

Another problem that occured with tissue as well as cells was the cryopreservation. If no reagent was added, there is easily RNA degradation happening even in samples shock frosted with liquid nitrogen. That was not true for primary cells in good condition or cell lines but for highly senescent and stressed cells. 

The other risky step is after the RNA precipitation / washing steps: If someone used the buffer or water you use for your RNA, the dedicated RNA pipettes were used somewhere else, the general RNA work place problems... and so on.

Also if you are running an agarose gel, are the buffers and systems RNAse free? We never had the problem that our RNA degraded during this step but we make sure that we clean the electrophoresis chambers, use RNAse free loading buffer and fresh TAE buffer (and filter tips of course for pipetting).

One site note: You really don't need to work on ice. Ice or ice water or the ice boxes actually can easily be the cause of your degraded RNA since it is so easy to get water droplets everywhere. The 4°C centrifugation step is enough and if you have a normal lab RT, nothing will happen to your RNA during your isolation. The RNase is your biggest enemy, not room temperature. If you still feel better with a cooled sample, use a cool rack that you can clean and that is dry during your isolation. 

Also if I look at the first picture: There is a little bit non-degraded RNA left. That is a hint for me that the problem probably occured before you added the reagent. Normally if you have a contamination problem afterwards, all is degraded. It doesn't need to be so, but this is where I would start to look for the problem.

 Hi claire, Thanks you very much for your suggestions.

The cells are triple negative breast cancer cells and they were harvested from a confluent 75ml flask with ice cold PBS and stored at -80. These cells were harvested the day before RNA extraction was attempted. 

Cells from 75 ml flask? I guess, I know your problem. It looks like a lysate excess. Twice or three (or even more) times dilute your cells (via x1 PBS) and then add TRIsure. Excess of cells means excess of nucleases and no guanidine salt helps in this case. Good luck!

What I dont understand is that i attempted the exact same method with the same cells with my supervisor and the RNA bands came out perfectly ( see attached). When i have tried the extraction myself i get the results originally posted. So im not sure if the dilution is the problem if there has been no variation in the method

Okay, if you didn't shock freeze your cells before you stored them at -80°C, than this would be the most possible cause for your problem. The freezing process down to -80°C is too slow and the RNA will start to degrade.

If you use your cells just for RNA isolation, I would recommend to use your reagent to directly lyse the cells in the flask and then freeze the cells/reagent at -80°C. It is really fast and easy, you'll save time and your RNA is safe.

Did you take an aliquote from the same stock of cells? 

Hi claire, i tried to isolate the rna again except this time i directly lysed the cells in the flask with TriSure with a succesful result. I think, as you described, the problem was that the RNA was already degraded from harvesting.