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DNA quantification: NanoDrop vs. Picogreen - measurement of ChIP Input DNA (Mar/16/2010 )

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!
Attached File

-NemaToStella-

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!!

-Pradeep Iyer-

Pradeep Iyer on Mar 17 2010, 07:11 AM said:

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.

-NemaToStella-

NemaToStella on Mar 18 2010, 03:02 PM said:

Pradeep Iyer on Mar 17 2010, 07:11 AM said:

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

-Pradeep Iyer-

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

-DocFlow-

yeah i agree!!! :D

-Pradeep Iyer-

Thank you guys so much!

-NemaToStella-

NemaToStella on Mar 19 2010, 09:34 PM said:

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.com/downloads/case-stu...0app%20note.pdf

-Olga Likhodi-

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.

-HomeBrew-

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.

-wdavis-