the room for RNA isolation is separate. Tanks are also separate for RNA. all the tips are properly treated with DEPC water, but still sometimes I am unable to visualize on gel, even if the concentration loaded is more than 50ng. I usually load 2 micro lit. of RNA with concentration above 50 ng per microliter?
Hi, are your tubes also RNase free? What agarose did you use? Cheers, Nadine
What are you using to stain the RNA? In my experience if it's Ethidium Bromide it may not be as sensitive as other reagents.
It just doesn't seem to be as sensitive for RNA as it is for DNA. If doing an RNA gel I normally use a product called Gelstar from Lonza, it's not done me wrong yet.
hi
treat your Agarose tank with SDS to remove any RNase - if you can load 1 ug it well be better
Hay hi
Ethidium staining is strongly enhanced by the double stranded structure of native DNA because it will go and intercalate. Staining of denatured, ssDNA or RNA is relatively insensitive, requiring some 10 fold more nucleic acid for equivalent detection. Another limitation is that the fluorescence of ethidium is quenched by polyacrylamide, reducing sensitivity by 10-20 fold in PAGE gels.Try with fresh EtBr,we are also using EtBr only.
i am not agree with Shaun, i always run the RNA on agarose containing ETBR, every time i see the bands, i think you should load more quantity about 5 microlitre, try it.
I also think that it is unlikely that you would not see RNA because it is single stranded unless you are deliberately doing something to denature it. Specifically tRNA and rRNA are not single stranded, but partially double stranded, although mRNA is sufficiently single stranded to stain less well. But unless you are running gels in alkaline conditions or with other denaturants you will still see staining if there is 50ng RNA there - so I think you have a problem with intact RNA yield. Oligonucleotides, free nucleotides and EDTA all have absorbance that you might be measuring if you are using spectrophotometry. Use a specific fluorescent dye system if you can.
Use fluorescent dye to measure RNA fluorometrically I mean, not just use it in the gel.
If you are running a formaldehyde-agarose gel for RNA, you will have to wash the formaldehyde away in order to visualize the RNA . Formaldehyde gives background fluorescence. And as others have mentioned RNA does not stain as well as DNA.
Christopher Blossey is not quite correct. Although tRNA , rRNA and mRNA have single stranded regions, all also have internal double helices, which in the case of tRNA and rRNA are a substantial part of the whole molecule, and all three do stain with ethidium bromide, although not quite as strongly as dsDNA. Gel systems for RNA sizing such as formaldehyde agarose do denature these regions and then EtBr staining is insensitive. In standard agarose, your size markers will be inaccurate for sizing the RNA (because it exists in a variety of complex structures) but it (the RNA) should stain reasonably well. But remember that mRNA is also a complex mixture of sizes, so it runs as a smear, without much RNA or staining at any given size. So if you have purified mRNA, rather than total RNA it lacks prominent bands and is hard to see. In this circumstance you will need to load more.
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