The non-denatured mRNA migrates in two forms: (i) as a higher order structure (due to secondary structures inherent to RNAs); this form seems to dominate; and (ii) as a form without these secondary structures formed. Denaturing then abolishes formation of the secondary structures, resulting in one band, which should correspond in size to the length of the mRNA.

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When you observe two peaks for undenatured mRNA on an agarose gel, it may be indicative of secondary structures or alternative conformations in the RNA molecules. RNA molecules are known to form various secondary structures, such as hairpins and stem-loops, due to base-pairing interactions within the molecule. These structures can affect the migration of the RNA on the gel, leading to the appearance of multiple bands.

Here are a few possible explanations for observing two peaks with undenatured mRNA on an agarose gel:

To confirm the nature of the observed bands, you may consider running denatured samples alongside undenatured samples. Denaturation involves disrupting secondary structures by heating the sample. If the multiple bands disappear or merge into a single band upon denaturation, it would support the hypothesis that the observed bands in the undenatured sample are due to secondary structures.

It's important to note that RNA is generally more prone to forming secondary structures compared to DNA. The denaturation step is often employed to disrupt these structures, providing a clearer representation of the molecular weight of the RNA on the gel. The presence of multiple bands in undenatured RNA samples is not uncommon and is often attributed to the structural complexity of RNA molecules.