RNA storage

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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RNA storage

Postby newbie17425 » Feb 05 2016 4:22 am

Hi,

I stored my RNA which I made around 7 months ago. The yield then was 850 ng/uL. Today, I needed to make cDNA out of the RNA so I measured it again to check the concentration of cDNA and surprisingly, the yield was down to 370 ng/uL.
Some points to be considered:

The RNA was eluted in 30 uL nuclease free water.
The first time I generated cDNA was within a day from the initial preparation of RNA.
The RNA was freeze-thawed only once and today the 2nd time.
The RNA was stored at -80 degrees
The 260/280-230 ratios didn't change much and still showed 2.00

What could be the cause of the decrease in yield over a period of time and what can be done to prevent this?

Thanks
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Re: RNA storage

Postby relaxin » Feb 05 2016 12:19 pm

Did you mix it well after thawing on ice?
The RNA could be degraded. If you need to use it again for other assays later, it is better to synthesize more cDNA, purify through spin column and store it as cDNA.
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Re: RNA storage

Postby newbie17425 » Feb 07 2016 1:17 pm

relaxin wrote:Did you mix it well after thawing on ice?
The RNA could be degraded. If you need to use it again for other assays later, it is better to synthesize more cDNA, purify through spin column and store it as cDNA.


Didn't mix it well though.
Is elution of RNA in water a problem?
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Re: RNA storage

Postby relaxin » Feb 07 2016 4:14 pm

Elution of RNA with water.

When you thaw your RNA, the RNA tends to be non-homogeneous. You need to mix well before pipetting. Otherwise, the O.D. reading will be way off.

This applies to any solution, reagent or buffer etc.
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Re: RNA storage

Postby newbie17425 » Feb 11 2016 7:52 am

relaxin wrote:Elution of RNA with water.

When you thaw your RNA, the RNA tends to be non-homogeneous. You need to mix well before pipetting. Otherwise, the O.D. reading will be way off.

This applies to any solution, reagent or buffer etc.


Sorry for my late reply. I did pipette up and down and it seem to have improved but only for a few samples. For others, the yield was lower which I guess is because of RNA degradation. What can I do to prevent RNA degradation?
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Re: RNA storage

Postby r.rosati » Feb 11 2016 10:44 am

newbie17425 wrote:Sorry for my late reply. I did pipette up and down and it seem to have improved but only for a few samples. For others, the yield was lower which I guess is because of RNA degradation. What can I do to prevent RNA degradation?


Degraded RNA absorbs as much as intact RNA, so this wouldn't explain the drop in absorbance.
Are you within the linear range of your instrument? Did you dilute the samples to read them back then, and not now?
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Re: RNA storage

Postby relaxin » Feb 11 2016 10:47 am

Pipetting up and down in small volume will not mix the RNA well. You need to mix with vortex mixer. That explains your inconsistent OD reading.

To prevent degradation of RNA, I used to treat my RNA with RNAsecure reagent and store it at -80 degree C.
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Re: RNA storage

Postby newbie17425 » Feb 11 2016 11:19 am

relaxin wrote:Pipetting up and down in small volume will not mix the RNA well. You need to mix with vortex mixer. That explains your inconsistent OD reading.

To prevent degradation of RNA, I used to treat my RNA with RNAsecure reagent and store it at -80 degree C.


Can I add RNAsecure now to my RNA samples to prevent further degradation?
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Re: RNA storage

Postby relaxin » Feb 12 2016 11:46 am

Yes, it is better late than never.
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Re: RNA storage

Postby nekameneka » May 06 2016 9:16 am

Changes in concentration on Nanodrop will not tell you if your RNA is degraded or not, concentration will be the same. I would assume something has happened to the measuring itself.
If you still think your RNA is degraded you can simply load it on EtBr agarose gel, degraded RNA will have a lot of smears. If it is not degraded you will see one clear band.
Hope you will find the answer. :D
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Re: RNA storage

Postby Joannajone » May 30 2016 7:20 am

In order to increase the storage stability of the RNA samples, You can dissolved it in deionized formamide at -80 degree C.
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