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Strange nanodrop readings after RNA isolation, strange solution - (Dec/20/2005 )

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Please think with me on this mysterie!

Right after RNA cleanup (RNeasy) I placed the (prokaryotic) RNA on ice, I homogenized and measured duplo's (the duplo's had the same results) on the nanodrop.

I had strange results like this sample:

RNA concentration: 486 ng/µl
A260/280: 1.10
A260/230: 1.31

I placed the samples in -80 overnight, thawed it on ice, homogenized and measured it again.

Same sample:

RNA concentration: 2032 ng/µl
A260/280: 2.21
A260/230: 2.43

Not only did the concentration rise by up 4 times, also the quality was much better. Note that this only happens with the samples that had bad ratio's the day before.

On the bioanalyzer these samples show good integrity and no degradation.

I would like to understand this so that I do not need to freeze samples just to get a concentration.

What could explain this?

-Jasper Bok-

Hi, Jasper Bok. It's strange...... I wonder would it just because of the contamination or error occurred while you were preparing the nanodrop for genquant?

-Angel1024-

this can also be due to dilution problems maybe the RNA had not completely dissolved in the TE prior to measurement and then after some time gives much better readings.

Ensure you have a homogenous sample prior to quant.

-scientist555-

QUOTE (scientist555 @ Dec 21 2005, 02:28 AM)
this can also be due to dilution problems maybe the RNA had not completely dissolved in the TE prior to measurement and then after some time gives much better readings.

Ensure you have a homogenous sample prior to quant.



Thanks for your answer!
I think dissolving the RNA has something to do with it.
Google result:"Partially dissolved RNA samples have an A260/280 ratio < 1.6".
My RNA is not in TE but in water. That should not have anything to do with it, right?

I am looking for another solution instead of freezing it overnight at -80.
It is also advised in some protocols to incubate for 10 minutes at 55 to 60°C to dissolve the RNA pellet. How does the heating affect the RNA? Would it not cause degradation?

I would like to hear your thoughts about this.

Jasper

-Jasper Bok-

I would never heat RNA to such temperatures. Chemical mediated degradation can occur if even small amounts of metal ions are present

It may be something to do with the fact your RNA is in water. To get accurate 260/280 ratios the solution should be >pH7

-John Buckels-

QUOTE (John Buckels @ Dec 23 2005, 02:02 PM)
I would never heat RNA to such temperatures. Chemical mediated degradation can occur if even small amounts of metal ions are present

It may be something to do with the fact your RNA is in water. To get accurate 260/280 ratios the solution should be >pH7


Thank you for your answer.

So, heating RNA is too risky. I won't try that then with the precious samples.

I am aware that water could result in inaccurate readings. But look at the results I had, pH doesn't change after freezing and thawing. Or does it?

Any other tips about dissolving RNA?

-Jasper Bok-

hi
i heat RNA samples at 55° for,10° in order to well resuspend them.
For ratio >1.65, extracted from trizol manual : RNA sample was diluted in water instead of TE prior to spectrophotometric analysis. Low ionic strength and low pH solutions increase absorbance at 280 nm .

but ratios for RNA >2.1 signifies contaminations.... It's quite frequent to obtain ratio >2 but this ratio is too high to give a purity info.

-fred_33-

To re-suspend my RNA I actually freeze at -80C overnight then thaw and vortex (rather than risk heating)

PS Your "2nd" round of ratios look fine for both 260/280 and 260/230

-John Buckels-

I just got ratios of RNA purity >2.1 (they ranged from 2 - 2.3) after RNeasy clean-up. Does that mean my samples are contaminated? What could the contaminates be, since I just did the column clean-up? How can I get rid of them?
Thanks!

-soluene-

QUOTE (fred_33 @ Jan 9 2006, 02:10 PM)
hi
i heat RNA samples at 55° for,10° in order to well resuspend them.
For ratio >1.65, extracted from trizol manual : RNA sample was diluted in water instead of TE prior to spectrophotometric analysis. Low ionic strength and low pH solutions increase absorbance at 280 nm .

but ratios for RNA >2.1 signifies contaminations.... It's quite frequent to obtain ratio >2 but this ratio is too high to give a purity info.


Hello Fred,

For purity and integrity I use the bioanalyzer. I attached the result for this sample to this post. Can you obtain information about contamination by looking at the results?
The RNA is extracted from cyanobacteria.

Thanks, Jasper

-Jasper Bok-

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