I have a RNA sample with 300 ng/uL of concentration and 260/280 as well as 260/230 ratios higher than 1.9. I ran an agarose gel to test the quality of my RNA sample and my loading buffer had a denaturing agent formamide. I followed the protocol mentioned in one of the articles about RNA electrophoresis. It says that I can run my RNA on agarose in normal TAE provided I have formamide in my loading buffer. However, I do not see any sort of band which might correspond to my RNA nor anything in the wells. What problem could this be that I am not being able to understand?
Add formamide in your agarose gel and rather using TAE, use FA gel buffer. Heat your RNA samples with RNA dye at 65 degree centigrade for 3 to 5 minutes and cool in ice then load on gel. First pre-run the gel with RNA dye only then load sample and run between 60 to 80V. Use 2-3 different concentration of RNA for loading.
We run our RNA samples on a denaturing Agarose gel containing Formaldehyde anf MOPS as running buffer. It gives nice results. see the link for detailed methd.
http://www.its.caltech.edu/~mirsky/labmanual/pdf/rnagel.pdf
best wishes
SD
You can also get a good idea of RNA quality by running it on a normal DNA gel, the same way you run DNA, as long as you don't need to see exact sizes.
But RNA can also degrade pretty fast if it is left out. How long did you wait between measuring the concentration and running the gel? Can you upload a photo of your gel? Can you see the ladder?
As long as you don't leave the RNA in the wells for too long, you shouldn't need to keep the apparatus or running buffer RNase-free. It works for me.
I use MOPS buffer but just put the formamide in the loading buffer not the running buffer which makes it a bit safer and works well. I also post stain the gels for half an hour and then destain in sterile water for half an hour which improves visualisation especially if concentrations of RNA are low. Do not run gels at too high a voltage as heat makes the RNA degrade faster so slow and cool better. If in a hurry RNA Reliant gels are good but expensive.
Hi,
I agree with Jacqueline and Ali, that you need controls to discard other issues with the electrophoresis, staining, etc. Can you see the DNA ladder or any control that you can load? How much RNA are you loading? It would be very useful if you upload a picture. If the RNA is degraded it can be seen in the gel. I run RNA gels in TAE and also MOPS/formamide and both work. The samples with denaturing conditions and heating so you can see the bands and their size, but even without you should be able to see something, like a smear, in the gel.
I agree with all the point above about cleanliness etc. RNA is very susceptible to degradation, and even when your rRNA bands look intact, your large mRNAs may well not be. But no-one seems to have mentioned yet that if you are using ethidium bromide as a nucleic acid stain and you are truly denaturing the RNA, as you will very likely be by heating with a formamide containing loading buffer, then it will stain very poorly, as EtBr is an intercalating agent requiring double stranded regions in the RNA to stain. Use an appropriate dye, such as one of the SYBR green II dyes with a formaldehyde gel, or, as these dyes are expensive, use less denaturing conditions prior to loading. Whatever you do, it may well be that the RNA is degraded (oligonucleotides will still give the same 260/280 ratio), so if possible check your extraction, loading and gel protocol with a test sample of a commercial RNA.
Thank you all for your answers! However, I can see the positive control RNA as well as the RNA ladder staining very bright. The problem seems to be only with my experimental sample which is hard to figure out. Any suggestions?
maybe you can try bleach gel..simplest denaturating gel
https://www.ncbi.nlm.nih.gov/pubmed/22222980
Hi Anand,
I am facing the same problem now. I get good RNA concentration about 300ng/uL with 260/280 ratio from 1.8-2. However, when I ran it on 1.5% agarose gel in 1X TBE with a positive control, the control can be observed but none of my samples are visible. No smear or any bands. Very clean lane. New gel and buffer was used.
You had mentioned the same problem a year back. How did you solve the issue? Any help would be appreciated.
P.s. Previously I have extracted RNA from cell cultures and ran it on the same gel composition. I never faced this issue.
Thanks,
Varsha
Varsha, what is your positive control? It is possible that your RNA concentration is not what you think it is.
mainly this was seen in fresh specimens, can be using RNase in your specimen treatment.
thanks a lot.
Hi Varsha,
I am confronting the same problem, the concentration is 540ng/ul with 260/280 ratio 1.76, but the ladder only could be seen in gel, how did you solve your problem?
Thanks
Reyhaneh
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