Dear colleagues,
Could you please suggest the best way to thaw and freeze purified mRNA in water.
I have batches of unencapsulated mRNA in nuclease-free water supernatant from nanoparticles and store it at -80C. I was thawing this mRNA supernatant for a few minutes on ice, but it took too long, so I then thawed it in my hand as recommendsd by lab colleagues. Upon quantification, I found that in 2/3 samples the concentration of mRNA that is way too high and can't be real. In 1/3 samples the concentration of unencapsulated mRNA was 0 ng/uL, which is great (all mRNA encapsulated into nanoparticles), but suspicious.
I suspect that when I was thawing the supernatants in my hand, the mRNA broke apart into smaller 4-6 fragments which made reading way higher.
It shouldn't be a problem, I thought. But it seems that quick thawing breaks mRNA apart.
There is no reason why quick thawing of mRNA should break it; furthermore, even if breakdown into smaller pieces does occur for any reason, it should have no influence on bulk quantitation based on spectrophotometric methods, be it by direct A260 absorbance or by indirect fluoroscopic measurement using a fluorophore like SybrGreen
I can't comment on your encapsulation or on the possible release of mRNA from the nanoparticles. But I was curious about the effect of freeze-thaw cycles on RNA integrity once, so I took samples of high-quality RNA (RIN 9) and freeze-thawed them repeatedly by transferring the tubes between my gloved hand and dry ice. After 5 freeze-thaws there was no difference and after 20 freeze-thaws I think it lost about 0.5 RIN. There's no way thawing your mRNA did that much damage to it, there must be some other explanation for the odd results you're getting. Perhaps the lipid nanoparticles do something weird when freeze-thawed? Do they aggregate during freezing?
@Laura Leighton,
Thank you for the suggestion. I thought it was damaged, but actually the volume of the reaction was 500 times smaller of what I thought, so not much mRNA was Lost and the numbers make sense.
Kind regards,
Maria
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