Nanodrop vs Fluorometer

[moerae]

Hi all,

I'm about to start using the SureSelect QXT kit as the DNA input is very low (50 ng). I talked to the technical specialist beforehand and he said that the DNA input has to be relatively exact (5 ng +/- is all right) is important for the shearing step.

We always measure our DNA concentration using the nanodrop and my samples are ~500 ng/uL. However, the fluoremeter readings are completely different from the nanodrop (~30-50% difference). I was wondering why this is the case? I will be running the samples on the gel to make sure it's not RNA or anything like that but if anyone has ideas what could be happening then that would be a great help.

[nucacidhunter]

NanoDrop measures absorption in different wavelengths and the 260 nm reading is used to calculate concentration of nucleic acids. Any nucleotide (single nucleotides, oligos, RNA and ds or ssDNA)contributes to the absorption. Therefore, the measured concentration is not exclusive to dsDNA fraction. On the contrary, fluorometric methods such Qubit or ds PicoGreen reagent measure only fluorescence from dsDNA. dsDNA is the only nucleic acid fraction that contributes to transposon based tagmentation such as QXT kit and other fractions that are taken into account with NanoDrop do not participate in reaction. The difference between NanoDrop and fluorometry depends on DNA extraction protocol and how efficient the used method was to extract dsDNA only. Protocols that use RNase solution to resuspend the pelleted DNA results in considerable over estimation because degraded RNA also will be measured by NanoDrop. I have seen over estimation by 10-15x with NanoDrop in comparison to PicoGreen. It is less likely to see nucleotides in a gel, so that may not help your QC.

[moerae]

Thanks for that. I'm just concerned if the nanodrop and fluorometer readings are very different and I need precisely 50 ng of DNA, I'll definitely go with the fluorometer reading; however, that would still be a lot of crap in the sample according to the nanodrop? I don't know if that makes sense...

[nucacidhunter]

Transposons usually are sensitive to inhibitors. You will get more consistent results if you clean your gDNA with Zymo gDNA clean and concentrator or 1.5x AMPure beads (for high throughput). With clean up step you can adjust your elution volume to obtain ~2x the required concentration and after a PicoGreen dilute it for required reaction concentration. The best buffer for elution would be Qiagen EB or TE0.1 pH 8.0 as EDTA in TE could inhibit tagmentation reaction (for Nextera not sure about transposon in QXT). Expected loss is 10-20%.

[moerae]

Ok. I don't have any gDNA clean up kits, but I do have AMPure beads. Do you by chance have the exact protocol for the clean up?

[moerae]

I also forgot to add that we use the Gentra Puregene blood kit to isolate DNA from blood with no RNAse treatment. Do you also think it would be wise to do an RNAse treatment?

[nucacidhunter]

I do not know about that particular DNA extraction kit, but generally commercial kits will have RNase or some other ways of removing RNA from extracts before resuspending DNA. You have mentioned that NanoDrop is estimating concentration ~2x of PicoGreen indicating that RNA has been removed. I would suggest to follow Beckman PCR clean up protocol (https://www.beckmancoulter.com/wsrpo...000387v001.pdf) on a less important sample for trial using less bead (1.5x vs 1.8x) to remove smaller fragments that will not contribute to your library.

[Diagnostics Joe]

I find the Nanodrop actually overestimates the DNA quantity a lot of the time at low concentrations and would recommend using the Qubit or other down at these levels. It'll take a while longer to generate the reading however. I've done a comparison here:
http://www.personalizedgenes.com/nanodrop-or-qubit/