All lanes were loaded with 5 µl sample material. Concentrations RNA increased per cell amount as determined by photometry. Ratio's were good. 2% non denaturing agarose gel was used (the 2% was a little mistake). RNA samples weren't heated prior to loading onto gel.
There are a couple reasons it could happen. One is if your RNA was degraded, but I don't think that is the case based on not seeing a lot of background smearing on your gel.
The other reason could be that you did not heat your sample and also ran a non-denaturing gel. RNA will form secondary structures that alter it's migration on a gel. Did you use loading dye with formamide? I generally heat my samples in formamide loading buffer before loading on a non-denaturing gel. I use non-denaturing gels just to get a quick sense of quality/quantity of RNA. If you want to have an accurate analysis of RNA on a gel you have to use a denaturing gel.
Finally, did you actually quantify your RNA? It looks like you have an increase in RNA as you increase the cells, but the amount of RNA (5S) does not appear to be doubling as would be expected. This could be because of slight variations in the extraction process or because of the gel issues mentioned above.
Kara A Boltz Thank you for your answer. Yes, I did quantify the RNA (see attachment). The goal of the gel run was indeed to get a quick sense of the quality and quantity of the RNA, and we are thought that non denaturing gels give results acceptable enough to do a quick semi- qualitative and quantitative analysis. I discussed with the project councellor and he told me that the band I thought was the 5S band, probably wasn't 5S but instead degraded RNA. That makes sense. He told me that I was able to run a proper PCR with this material nonetheless, but that I should first check the RNA integrity using bioanalyser to confirm the conclusion drawn from gel analysis (i.e. degraded RNA).
- I agree with your supervisor although Kara makes some excellent points
- where PCR is concerned, as long as you can see integral 18s and 28s subunit bands your PCR is sufficiently undegraded that your PCR will work fine
- I have PCRd from RNA that looks like yours and indeed worse and it woeks well for band identification
- It is only when performing procedures like micro arrays or qPCR that you need to pay closer attention to the S:N in your RNA
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