The easiest solution is the 1-1.5% agarose gel. If you see two nice bands of rRNAs (28S and 18S) , with the upper one being more brilliant than the lower ons you can "assume" that your RNA is OK. Extra bands among these, or a faint 28S band indicates degradation. If your downstream applications require extra-quality RNA then go to the Bioanalyzer or the denaturing gel.
The easiest solution is the 1-1.5% agarose gel. If you see two nice bands of rRNAs (28S and 18S) , with the upper one being more brilliant than the lower ons you can "assume" that your RNA is OK. Extra bands among these, or a faint 28S band indicates degradation. If your downstream applications require extra-quality RNA then go to the Bioanalyzer or the denaturing gel.
See, if you want to check the integrity of RNA, then you should go for denaturing gel. Formaldehyde is in the MOPS buffer as well as in the gel mix for RNA. Formamide is added in the loading dye for RNA and it is important. Formamide and Formaldehyde have different roles to play. Formamide is used as an RNA stabiliser in gel by deionizing RNA whereas formaldehyde is to prevent secondary structure formation in RNA.
For checking integrity.. 1. check by spectrophotometer for 260/280, i always y run a denaturing formaldehyde agarose gel. 2:1 ratio of 28S: 18S is a good indication that the RNA completely intact
If you want to avoid formaldehyde (because you do not have it), you can also do a polyacrylamide-urea gel. This is also denaturing and gives you very nice bands. It doesnt show you the larger bands very well, though. You could try 6%.
Tell me if you need the protocol.
Cheers, Uli
If you have access to a bioanalyzer (Agilent), that would be the best option - expensive, but seeming to become the new standard for assessing RNA integrity.
1X TBE agarose gel (0.8 % agarose) could roughly allow you to appreciate integrity by comparing ribosomal band intensities. The procedure is inexpensive and time saving.
I agree to Antonios comment. Its easy to jsut run a agarose gel 1-2% would give you a rough estimate of RNA integrity. I have seen that RNA with bad bioanalyzer results show up as smear in an agarose gel.
Hi there, i would go with Angela Orshinsky and strongly recommed you to use bioanalyzer (Agilent) you will only use very small amount of your RNA, you will be able to maintain it, and will have the most accurate determination for your RNA integrity ever. Best
If you are going for quality and integrity then go for non-denaturing gel, but if you are going for northern analysis or another thing go for denaturing gels.
The easiest solution is the 1-1.5% agarose gel. If you see two nice bands of rRNAs (28S and 18S) , with the upper one being more brilliant than the lower ons you can "assume" that your RNA is OK. Extra bands among these, or a faint 28S band indicates degradation. If your downstream applications require extra-quality RNA then go to the Bioanalyzer or the denaturing gel.
I'm just wondering about Himanshi's comment. It seems as if the web is full of citations referring to formamide as a denaturant for RNA. A typical quote would be: " Formamide and urea are the two most common agents which accomplish chemical denaturation. These substances act to disrupt the hydrogen bonding between the nitrogen bases, thereby removing the effects of base pairing." Which to me seems to be synonymous to "preventing secondary structure". I'd be curious to other comments on the roles of formamide vs formaldehyde - or a bit of eloboration on this from Himanshi, of course. Thanks!
You can use either one but in non denaturing gel you have much higher chances of RNA hydrolysis. Therefore you have to take extra precautions to avoid RNase contamination and RNA degradation.
You may use 1X TAE buffer containing 0.1 % DEPC treated water. Whereas, for denaturing or non-denaturing RNA they gave almost the same result.
Personal opinion; I prepare to denature the RNA before running on 1-1.2% agarose and special gel cast, tray etc.
Has anyone ever tested denaturing vs native head to head on the same samples for RNA integrity? Any images to share?
Another very relevant Researchgate thread is at:
https://www.researchgate.net/post/Can_anyone_help_me_with_gel_electrophoresis_of_total_RNA?view=5ad0f9c9b0366dcf7158e432
Just use formamide (secondary structure reliever) in your sample with loading dye/you can use urea instead of formamide>heat at 95 C>chill on ice and load. MOPS buffer is not denaturing substance here, it just maintain pH around 7 only. Formaldehyde here denatures RNA..
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA