Why do I got 3 bands of RNA gel? - (Jul/28/2007 )

I have just run a RNA agrose gel, but I find 3 sharp band on the gel. Does anybody have the same experience? I know it should be two bands. Why it give me 3? Thanks.

Total RNA is extracted from human lymphoctes line(GM cells).

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Where exactly are these bands? When I run a regular DNA marker with the RNAs (w/o denaturing) on a 1% agarose gel, 28S looks like 1.5 kb and 18S like 750 bp. Is your third band higher or lower than any of them?

In fact, eukaryotic total RNA has 4 major rRNA species, 28S, 18S, 5.8S and 5S. The latter two are less than 250 bp, so some RNA isolation kits such as Qiagen RNeasy do not elute them. (Their columns bind RNA longer than 250 bp). In such cases you don't see those bands but you definitely see 28S and 18S.

On the other hand, there are some abundant mRNAs in the cytoplasm. Although you don't see them as "shiny" as the rRNAs, they can also give you sharp bands.

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Can it be DNA? Perhaps you have both DNA and RNA. It is not uncommon to have both in your sample after RNA extraction.

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you may have DNA and then 28S and 18S
Or you have 28S 18S and 5S RNAs
May you attach a picture?

5S is a tiny band at end of gel in general cases, and DNA behave itself as a big band in upper part of the gel, making kind of a smear.
If you column-extracted the RNA, 5S goes in the flow through so you'll gonna have DNA in your sample.

Did you denature enough your RNA sample ?

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