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DNA quantification: NanoDrop vs. Picogreen


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#1 NemaToStella

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Posted 16 March 2010 - 03:15 PM

Hello,

I pretty confident that there is someone out there who can help me: I am doing ChIP-seq using different sources of chromatin (cell lines and whole animals) and recently encountered a problem with the quantification of input DNA which will be used to make sequencing libraries...

If I measure the (reverse-crosslinked/RNaseA and proteinase K digested/chloroform-phenol purified) input DNA using the Nanodrop it gives me a reading of 200 ng/ul (A260/280=1.93, A260/230=1.86). If I measure the DNA concentration using Invitrogen's PicoGreen Assay I get 48 ng/ul for the same sample! I've used the NEB 100 bp ladder DNA as standard (using 1000 / 100 / 25 / 10 / 2.5 and 0.25 ng/ml).
From the RFU readings of the fluorometer I calculate slope and intercept of the calibration line which allows me to calculate the concentration of my samples. This works beatifully for the RFU readings of the standards.

Does anyone happen to know why I am getting these weird (~ 5-fold different) readings? I am starting to think that there might be a mistake in the dilution of my standards...

Thanks so much in advance!

#2 Prep!

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Posted 17 March 2010 - 04:11 AM

Well I am not sure of 5 fold difference, but the Picogreen dye is specific for double stranded DNA, so if your system is accurate and precise, I would rather rely on the Picogreen values than the OD ratios. You must have read the insert for the duye and it claims that even more than a 1000 fold RNA or ss DNA wont alter the efficiency of the dye, and with my experience in Picogreen, I would say that is the best bet.
The only thing tat i wud cross chek is the reliability of the 5 time less reading.
I will prefer using the same standards as your sample DNA. This can give variations in reading. As u have used a ladder, the binding to different base pairs length at different concentrations may vary so I sugggest you use the same standard DNA as your DNA in question!!

Best luck!!
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#3 NemaToStella

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Posted 18 March 2010 - 12:32 AM

Well I am not sure of 5 fold difference, but the Picogreen dye is specific for double stranded DNA, so if your system is accurate and precise, I would rather rely on the Picogreen values than the OD ratios. You must have read the insert for the duye and it claims that even more than a 1000 fold RNA or ss DNA wont alter the efficiency of the dye, and with my experience in Picogreen, I would say that is the best bet.
The only thing tat i wud cross chek is the reliability of the 5 time less reading.
I will prefer using the same standards as your sample DNA. This can give variations in reading. As u have used a ladder, the binding to different base pairs length at different concentrations may vary so I sugggest you use the same standard DNA as your DNA in question!!

Best luck!!


Thanks for your reply! I'll trust the PicoGreen then.

#4 Prep!

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Posted 18 March 2010 - 12:50 AM

Well I am not sure of 5 fold difference, but the Picogreen dye is specific for double stranded DNA, so if your system is accurate and precise, I would rather rely on the Picogreen values than the OD ratios. You must have read the insert for the duye and it claims that even more than a 1000 fold RNA or ss DNA wont alter the efficiency of the dye, and with my experience in Picogreen, I would say that is the best bet.
The only thing tat i wud cross chek is the reliability of the 5 time less reading.
I will prefer using the same standards as your sample DNA. This can give variations in reading. As u have used a ladder, the binding to different base pairs length at different concentrations may vary so I sugggest you use the same standard DNA as your DNA in question!!

Best luck!!


Thanks for your reply! I'll trust the PicoGreen then.


Best luck then!!! :P
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Cheers!!!

#5 DocFlow

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Posted 18 March 2010 - 03:06 AM

this is the same problem we encounter here at our lab. We measure our DNA on the nanodrop and compared to picogreen UV is usually 3-30% higher than dsDNA. We've even seen a 10 fold difference, but that is a long time ago. I don't automatically think that one or the other method is superior and gives "more accurate" results, because both methods have pros and cons. The major con for picogreen is the use of a standard.
So my thoughts on this is that as long as you use the same method for measurements of all your samples it'll be all good

#6 Prep!

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Posted 18 March 2010 - 03:49 AM

yeah i agree!!! :D
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#7 NemaToStella

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Posted 19 March 2010 - 08:34 PM

Thank you guys so much!

#8 Olga Likhodi

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Posted 14 May 2010 - 08:32 AM

Thank you guys so much!


If the PicoGreen and NanoDrop readings do not
correlate, the NanoDrop readings are usually higher than
PicoGreen, indicating that the DNA sample may contain a
mixture of double- and single-stranded DNA, contaminants
which scatter light, or UV-absorbing materials that are not
nucleic acids.
From here: http://www.genvault......0app note.pdf

#9 HomeBrew

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Posted 14 May 2010 - 01:56 PM

I agree with Olga -- the two methods are measuring different things. One, the Picogreen, is measuring the amount of dsDNA in the sample, while the other (Nanodrop) is measuring anything in the sample that scatters or absorbs light at the wavelength used for the measurement.

From the Nanodrop literature:

Absorbance measurements made on a spectrophotometer, including any Thermo Scientific NanoDrop Spectrophotometer, will include the absorbance of all molecules in the sample that absorb at the wavelength of interest. Since nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, they will contribute to the total absorbance of the sample.



#10 wdavis

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Posted 18 October 2010 - 12:25 PM

Has anyone had the reverse issue, with their Picogreen results being higher than their Nanodrop results ? They are not significantly higher but they definetly tend to be higher.




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