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RNA concentration given by Nanodrop

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9 replies to this topic

#1 xxcici

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Posted 07 January 2014 - 12:00 PM

Hi, I have a question about the concentration of RNA given by Nanodrop. I tested my RNA treated with the following methods: 1. immediate after extraction using kit; 2. stored at -80C for several days, thawed on ice; 3. stored at -80C for several days, thawed on ice and heated at 95C for 3 min, followed with chilled on ice. Then after testing by Nanodrop, the concentration of RNA is 4 ng/uL, 4.1ng/uL, and 8.9ng/uL. Why the concentration of RNA is improved so much after heating treatment? Which concentration given by Nanodrop is the most accurate one? 



#2 bob1

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Posted 07 January 2014 - 12:03 PM

At those sorts of concentrations the nanodrop (or any other method for that matter) is not reliable as tiny changes in the A260 or A280 will dramatically alter the concentration.



#3 xxcici

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Posted 07 January 2014 - 01:18 PM

Thanks Bob. Actually, I meet with this situation twice. So, I think heating must have some effect on the concentration of RNA. If I say heating makes RNA molecules dissolved better is the reason for higher given concentration, then, how we can usually test the RNA concentration by Nanodrop? We do not need to use heating every time. 



#4 bob1

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Posted 07 January 2014 - 07:03 PM

How was the RNA prepared?  If you did a classic phenol:chloroform extraction (e.g. trizol) then the RNA is precipitated and needs to be dissolved and you might be seeing an effect there if you froze the RNA before it was completely dissolved (most people heat to 55 C for a few minutes to ensure that the RNA is dissolved).  If the RNA was over-dried before dissolving it can be hard to dissolve and heating will help to dissolve it.

 

RNA that is dissolved already when frozen is not re-dissolved when thawed (i.e. it doesn't precipitate with freezing in the absence of salts and alcohols).  The heating may be doing something, but with such low concentrations ANY change will show up dramatically.  How do your RNA curves look?  If they are nice smooth curves and don't have a peak in the 230 nm range, then I would be much more willing to accept your premise.

 

How were the concentrations before it was frozen? Twice could just mean that you somehow picked up a tube with a higher concentration twice.  Many more replicates needed!

 

If there is anything to your theory, then you should be able to take the tubes thawed on ice and heat it to 95, cool and re-measure and see the same effect (to be completely accurate you would need to re-freeze it first). 



#5 phage434

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Posted 08 January 2014 - 07:08 AM

I find it remarkable that your RNA concentrations are so low. A typical prep makes micrograms of RNA. It suggests to me that your preparation is not working. As Bob1 said, the Nanodrop is highly unreliable for samples less than about 10-20 ng/ul.



#6 xxcici

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Posted 08 January 2014 - 06:17 PM

Thanks guys for your detailed explanation.

 

The RNA is extracted using QIAGEN column and eluted with water. The concentration of RNA is very low because the target in the sample is low. After RNA is eluted with water and before freezing, the concentration is 3-4 ng/uL. After freezing and thawing on ice, the concentration of RNA is similar as before freezing. Then, after thawing on ice and heating at 95C for 3 min, then on ice. The concentration is improved. 

 

This phenomenon was also found for my other sample RNA with higher concentration. Do you have some documents or papers reported that" Nanodrop is highly unreliable for samples less than about 10-20 ng/ul"? If I can get them, that would be perfect. 

 

Thank you very much again. 



#7 mdfenko

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Posted 09 January 2014 - 05:07 AM

are you sure that the volume of the sample is unchanged after heating?


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#8 phage434

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Posted 09 January 2014 - 11:09 AM

I'd recommend that you dilute some DNA samples down to 1, 2, 4, and 8 ng/ul and measure each several times on your nanodrop. You'll see what I'm talking about, and won't need a paper. Or, you can establish some protocols that work for you.



#9 Adrian K

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Posted 15 January 2014 - 12:01 AM

Thanks guys for your detailed explanation.

 

The RNA is extracted using QIAGEN column and eluted with water. The concentration of RNA is very low because the target in the sample is low. After RNA is eluted with water and before freezing, the concentration is 3-4 ng/uL. After freezing and thawing on ice, the concentration of RNA is similar as before freezing. Then, after thawing on ice and heating at 95C for 3 min, then on ice. The concentration is improved. 

 

This phenomenon was also found for my other sample RNA with higher concentration. Do you have some documents or papers reported that" Nanodrop is highly unreliable for samples less than about 10-20 ng/ul"? If I can get them, that would be perfect. 

 

Thank you very much again. 

 

Google and found this paper for you:

http://www.gene-quan...cation-2009.pdf

 

tl;dr

 

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#10 xxcici

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Posted 16 January 2014 - 09:42 AM

are you sure that the volume of the sample is unchanged after heating?

The volume should not be changed because heating and centrifugation is used. 







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