HI everyone, I just isolated my RNA from directly from blood by TRIZOL method, I got purity of 1.97 (260/280) which is pretty in acceptable region, but when I run my RNA on gel it shows 3 bands. as i know the bands will be 28s, 18s and 5s. so their band size will be as accordingly. but in this picture 18s shows double band size than 28s.
If the level of fluorescence of the 18s band is equal to that of the 28s it means that the RNA is somewhat degraded. That won't affect the 260/280 ratio since that is a measure of protein contamination, not "intactness". Without knowing what the source of the RNA is, and how it was extracted, it's hard to make any guess as to the reason for the degradation. If you were to do a densitometer tracing of your gel the 28s:18s ratio should be 2:1, which is the benchmark for intact RNA.
Just a short question: did you use a denaturating agarose gel (e. g., containing formaldehyde)? And did you used agents like formamide/formaldehyde (+ heating to 65 °C) when you prepared your samples for loading onto the gel?
If not then the bands you see could be due to the formation of secondary structures. RNA as a single stranded nucleic acid is prone to form these if not denatured.
Regards, Christian
Hi Christian,
While I agree that the use of denaturing agarose gels, and pretreatment for removal of secondary structure is excellent practice for running RNA gels, that wouldn't affect the migration of the ribosomal RNA bands to any appreciable extent, nor would it affect intensity of fluorescence of those bands. Having run probably thousands of such gels the image that was shown still demonstrates some degree of RNA degradation.
Regards,
Ted
Hi Ted,
you are right. So first the problem of degradation has to be solved. Then it is recommended to use a denaturating agarose gel for checking the samples.
Cheers, Christian
Hello TEd
i was isolating total RNA from blood from TRIzol method,
i just got 1.97 purity, with 3 visible intact bands , the problem is that the band intensity is not same as i find normally in other rna gel.
Hi Christian.
you are right that we can used a denaturing gel for RNA gel, and i did it o time ago, when i got a single band on RNA gel, but i mix MY RN with formamide i got 3 band s.
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