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Author Topic: Shrinking mRNA Peaks over time in CE  (Read 1295 times)

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jeffmoonchop

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Shrinking mRNA Peaks over time in CE
« on: November 23, 2018, 08:07:03 AM »

Hi all, I'm testing the stability of mRNA by CE over time. The mRNA is encapsulated in lipid nanoparticles so when I prepare the samples I break down the particles to release the mRNA using Triton. When I only just make the particles the mRNA purity is high at the same as the standard (usually around 93%), but over time the purity decreases as expected especially at 25C conditions. For example, it reduces to 50% after three months.

The thing is by normal degradation wouldn't you expect other peaks to appear if the mRNA strand is hydrolysing or breaking up?

What I see is no extra peaks but the peak area/height just reduces. Before the peak there is always a front or hump, That front doesn't change but because the peak height changes the ratio of peak areas reduces compared to the front.

My question is, what is happening to the mRNA? Is it just evaporating or is it being bound more strongly by the lipid over time so it's not being released? Does anyone have an idea and how I could test it to confirm what is happening?

many thanks
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Babcock_Hall

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Re: Shrinking mRNA Peaks over time in CE
« Reply #1 on: November 24, 2018, 01:53:15 AM »

RNA is subject to a number of degradation reactions, including transphosphorylation.  Ronald Breaker has studied transphosphorylation.  I am at a loss to explain why other peaks do not show up.
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Yggdrasil

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Re: Shrinking mRNA Peaks over time in CE
« Reply #2 on: November 26, 2018, 10:39:39 AM »

RNA can be hydrolyzed down to single nucleotides, which likely are too small to be seen in the capillary electrophoresis (e.g. it looks like the LM marker corresponds to 15 nt).  Furthermore, if the nucleotides are dephosphorylated to nucleosides, they would not be seen by electrophoresis since they do not have any charge.  Single nucleotides or small oligonucleotides may also not be detected by the CE instrument depending on the detection method used (e.g. CE instruments commonly use fluorescent dyes that bind to nucleic acids, and these dyes will not efficiently bind single nucleotides or very short oligonucleotides).
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jeffmoonchop

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Re: Shrinking mRNA Peaks over time in CE
« Reply #3 on: November 26, 2018, 12:23:16 PM »

great thanks for that answer. I'll look more into those degradation mechanisms. I'm not a biologist so its great help.
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jeffmoonchop

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Re: Shrinking mRNA Peaks over time in CE
« Reply #4 on: November 26, 2018, 01:36:33 PM »

Would you be able to offer a way to stabilise mRNA to prevent degradation? I've looked at a few papers and they all seem to be enzyme based but I'm treating the mRNA like an API, are there any chemical additives I could use?

I'm planning on using antioxidants such as vit C and E in combination to scavenge oxygen radicals. Do you have any other ideas?
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Babcock_Hall

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Re: Shrinking mRNA Peaks over time in CE
« Reply #5 on: November 27, 2018, 08:43:37 AM »

Avoid contamination from RNase A.  There is a branch of quantitative polymerase chain reaction chemistry that deals with mRNA.  IIRC it is sometimes called RT-qPCR, where RT stands for reverse transcriptase.  I would look into this literature to see what things are recommended; Stephen Bustin is one name to look for.
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hypervalent_iodine

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Re: Shrinking mRNA Peaks over time in CE
« Reply #6 on: November 27, 2018, 05:46:35 PM »

Avoid contamination from RNase A.  There is a branch of quantitative polymerase chain reaction chemistry that deals with mRNA.  IIRC it is sometimes called RT-qPCR, where RT stands for reverse transcriptase.  I would look into this literature to see what things are recommended; Stephen Bustin is one name to look for.

RT-PCR is correct. You can also use other methods for quantifying and checking purity, but these rely on having expensive pieces of equipment. So does RT-PCR I suppose, but you're more likely to have access to something capable of that than you are a BioAnalyzer.

OP: to address your last post, there are plenty of ways to do ensure sample integrity, and they all involve a great deal of care on your part in handling the samples. As you know, RNA is not super stable, and so the first things you need to make sure you are doing is a.) using RNAse free solvents, vessels, and consumables (including RNAse free sterile filter tips for any pipetting); b.) working with your samples on ice and as sparingly as possible; and c.) storing them in a -80oC freezer. For storage and use, having your mRNA in 0.1 M EDTA solution (in RNAse free water) is good; you can also use plain RNAse free water, but you won't get as much mileage out of it, especially over a time frame of 3 months. Generally speaking, mRNA is best kept below pH of 5. Longer term you would want to use an acetate-alcohol based precipitation method (this is easy to look up).

Have a look at suppliers websites for other tips on this, they are usually pretty detailed with this sort of stuff. Thermo has some good information for example: https://www.thermofisher.com/au/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/working-with-rna.html 
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jeffmoonchop

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Re: Shrinking mRNA Peaks over time in CE
« Reply #7 on: November 28, 2018, 06:38:00 AM »

Thanks for the tips. I do use RNAse free stuff mostly working in a biological safety cabinet, and everything I use is one-time use sterile equipment etc. Some of the suggestions I've tried, but using EDTA increases the lipid nanoparticle size which isn't desirable. I need to balance the properties of the particle with the RNA integrity. At the moment I have a good method of stabilising the particles, but if I change something like the pH to stabilise the mRNA, the particle grows or doesn't encapsulate the mRNA.

My samples are stored at -80, -20 and accelerated deg at 2-8C, but trying to get it stable at -20. I also lyophilise the samples but they are less stable.
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