Simple answer - buy a Qubit or similar (ideally both). I now only use a Nanodrop for a quick check for DNA but mainly for the 260/280. It's also good for working out if you've got contaminants (i.e. phenol, ethanol). To answer Robert Cox's question, theoretically the dye in the Qubit kit should only bind to dsDNA so yes, it should be able to measure in the presence of NTPs.

The nanodrop is poor in my experience at quantifying

Devices using DNA binding dyes such as the qubit measure DNA concentrations directly, whereas the nanodrop quantifies anything that fluoresces at the approximate wavelength of DNA.

I compared nanodrop to picogreen when quantifying a probe generated for southern blotting and found the nanodrop concentration were around 40% higher. I would presume that this would be the case with qubit dye which is a similar chemistry.

You can buy a qubit kit to quantify low concentrations, it can easily detect sub nanogram concentrations down to around 0.1ng/uL

First of all, this depends whether your Nanodrop is using a fluorescent assay (ND3300) or absorption (ND1000/2000). For higher concentrations, absorption might be easier, but this has limited sensitivity.

For low concentrations, any fluorophore-based method is suited better due to higher sensitivity. Here, a thorough calibration will yield better results. I am not entirely sure whether Nanodrop (for fluorescence) or Qubit is actually superior.

How does the higher precision correlate with a higher price? Once you have bought the Nanodrop you do not have any further costs (in case you measure fluorescence). But if you always have to buy this substance for Qubit how much do you have to pay additionally per measurement?

We have all but stopped using our NanoDrop since getting a Qubit. NanoDrop certainly has a place in measuring purity (260/280 and 260/230) which the Qubit cannot do, however, I do not trust to accurately determine of nucleic acid concentration.

Spectrophotmetric methods do not measure nucleic acids directly, but the absorbance of whatever is in your tube. We use the qubit for measuring dsDNA, which it does by measuring the fluorescence of a double stranded DNA specific dye. Therefore, if there is protein, RNA or nucleotides in the sample, they are not going to significantly contribute to the measurement as they would with the nano drop.

We have found up to a 10-fold overestimation using the NanoDrop. That is not really a problem for cloning for example, but for NGS, it can be a critical and very expensive difference. Moreover, I wouldn't trust the Nanodrop at all under 10 ng/ul, where the Qubit handles sub-ng/ul fine.

Both machines have their place in a lab, but for DNA concentration, i'd go for the Qubit.

Can the qubit measure nucleic acid concentration in the presence of NTPs?

Simple answer - buy a Qubit or similar (ideally both). I now only use a Nanodrop for a quick check for DNA but mainly for the 260/280. It's also good for working out if you've got contaminants (i.e. phenol, ethanol). To answer Robert Cox's question, theoretically the dye in the Qubit kit should only bind to dsDNA so yes, it should be able to measure in the presence of NTPs.

The nanodrop is poor in my experience at quantifying

I have never used Qubit so can not comment on that, but as for Nanodrop...well it has some issues concerning accuracy (and I can tell it by trying at least 3 different Nanodrops in 3 Different laboratories in 2 countries). Problems with accuracy are especially visible while measuring low volumes or low quantities of nucleic acids. On one hand speaking of volume in troubleshoot they claim that evaporation of your sample can occur and this can distort the results, on the other hand I am not too convinced with that. So while using Nanodrop one should never trust a single measurement, and perform it at least twice (or better trice), but in this case if your sample size is only 20 uL and you use 15-20 % of it just for measuring quantity, sometimes you end up with amount of nucleic acid that is not enough to perform further procedures..so to sum up, if low volumes (10-20 uL) or low quantity ( less than 20-40ng/uL) of nucleic acid are not employed in your procedures go for Nanodrop, otherwise you should consider using more accurate methods.

I can say nothing about Qubit, but we have Nanodrop in our lab and don't use it. As the results are very nice reproducible (both for the same sample re-measurements and in dilution series) it is not clear what is measured indeed. The results seem to be greatly overestimated for double-stranged DNA. Also, we had no any advantages when tried to use the measurement result for PCR estimating.

I compared purified, size selected (1.5-2kb fragments) gDNA across 3 machines; NanoDrop, Qubit, Bioanalzyer. The nanodrop gave consistently 3.5-3x higher concentration readings than Qubit and Bioanalyzer, which were comparable with ~5ng. I believe the Nanodrop's accuracy (repeatability) is good but it's precision is far too off to be of use in NGS library prep, and, my opinion, qPCR.

Correction. I meant to say a 10.5-10x difference between nanodrop and qubit/bioanalzyer, not 3.5-3x.

I have just purchased a Qubit (not yet arrived), and will compare it to the Nanodrop. On comparing the Nanodrop to our plate reader (Multiskan Go), I tend to agree with James. The Nanodrop seems to over-estimate, especially at higher concentrations. We've noticed the following recently. Following a DNA extraction, the DNA was diluted 1/50 for measurement in the plate reader, or read directly with the Nanodrop. The plate reader gave a reading of 1.3 ug/uL, whereas the Nanodrop gave a reading of 3.5 ug/uL. Now here is the interesting part: If we measure the diluted sample with the Nanodrop, it also reads 1.3 ug/uL! I think that the Nanodrop's range for linearity is simply much smaller than advertised.

For NGS purposes, only use Qubit/Picogreen of even better qPCR to quantify the final libraries. Use Nanodrop to check for impurities, but neglect the concentration measurements. To our findings, Nanodrop consistently overestimates the other quantification methods by 2.5-3x in a (10-150ng/µl] range.

I had serious trouble deciding between qubit and nano drop. Your responses have resolved my doubts. In fact the best it is good to have both qubit and nano drop.

In our case the majority of DNA samples had less than 50ng/µl and generally the data of Qubit is better to define the dilution for PCR

Thank you guys. We have to decide whch one to buy..I think the winner is Qubit. Cheaper and probably as good as Nanodrop

Hi,

I'm doing RNA extraction from serum and CSF using mirVana PARIS and the concentration measured with Nanodrop is around 8 ng/ul. It's ok considering these fluids but when I use Qubit 1.0, the RNA is not detected (< 20ng/ml). It is possible that I'm obtaining no RNA?? Qubit should detect even in low concentrations.

Anyone else had this problem? 

Thanks!!

I would give a look to the Trinean's instruments. They have this new technology which allows you to measure 96 samples in 6 minutes without any dyes, and still get Qubit accuracy.

It's quite new but still worth to try it.

Hi,

I extract RNA from  plants and was using Qubit but planning to get a nanodrop to be able to meassure 230/260  ratio also for savings (no cuvets).

Thanks

Hello! thank you for this useful thread: these issues were driving me mad...depending on which one nanodrop or Qubit i was using i was from euphoria (detecting nice quantities of DNA extracted) to frustration (the Qubit was showing that i had extracted almost nothing at all!! so i had to start all from the beginning ) . Now i know what was this all about and for next time i would make the choice wiserly. Thank you

Comparison of Qubit:NanoDrop? Different purposes!

I am still starting the Qubit measurements, because I want to precisely measure my RNA concentration in samples. The problem with the NanoDrop is that is only capable of measuring nucleic acids, which is including DNA contamination and also free nucleotides, both of which I want to exclude. The Qubit promises to measure specifically RNA, without DNA and also free nucleotides. Additionally more precisely and with a much lower limit than the NanoDrop.

Good luck and bests,

Christopher

Update: Ok, so, interestingly, the Qubit gave me EXACTLY the same concentration as the NanoDrop did.. (we have in-vitro-transcribed RNA). No DNA contamination, no free nucleotides, however, two RNAs of exactly the same concentration look different (roughly 2-fold) on an agarose gel. But that's not a matter of this discussion here..

Hi everybody, for the newsfeed could get the information that maybe another machine (not nanodrop) will have less accuracy, but even if you know scandrop? Is scandrop similar to nanodrop?

I also had big differences when comparing bioanalyzer, nanodrop and Qubit. for RNA quantification.

Let me ask a related question: Can you obtain good 260/280 and 260/230 ratios with low concentrations of RNA? (about 100ng/uL)

I used RNeasy Micro Kit and these ratios are really bad in nandodrop measures.

Many DNA/RNA purification kits use solutions containing guanidine salts that have a strong and broad absorbance at ~230 nm even at low concentrations.

If you have some carryover of such salt, and if the concentration of your DNA/RNA is low, you cannot obtain correct readings using Nanodrop, because of the absorbance of the guanidine salts.

Trinean's DropSense instruments allow to determine dsDNA concentrations ranging from 1.5 ng/ul - 10000 ng/ul without the use of any dyes or dilutions. Furthermore, software (i.e., cDrop) is available that distinguishes absorbing buffer components (e.g. guanidine salts, phenol, ...) from the DNA present in your samples allowing a more accurate concentration determination. In addition to DNA, these instruments also quantify and purity check RNA and protein samples. Feel free to have a look at www.trinean.com 

Why not consider a Colibri: http://www.titertek-berthold.com/microvolume-spectrometers/details/product/colibri/action/show.html? This instrument is more accurate than anything else on the market. You are welcome to demo one from Thistle Scientific if you wish.

Fransesca; what is your endpoint application to use the RNA ?  For Microarray, we quantitate ONLY by Bioanalyzer although some literature says Qibit is fine, too.   For qPCR use the nano drop.  The caveat for nano drop is is quantitates ALL nucleic acids in your droplet, so if you have not done a step to rid your sample of DNA pre-RNA isolation (highly recommended) the reading will be mostly DNA.  

Look for DS-11 FX Series Spectrophotometer / Fluorometer by DeNovix Inc. The equipment combines fluorescence and absorbance capability in a single instrument.

Hi, I´m using Qubit to quantify RNA and also DNA for NGS. I read in the guideline of HS RNA measurement that to determine the purity of your sample, I can use the Qubit® RNA HS Assay Kit together with the Qubit® dsDNA HS Assay Kit, and that these measurements give you a much better

indication of sample purity than that produced by measuring the A260/A280 ratio.

My question is, once I have both values measured by Qubit (RNA concentration and DNA concentration), how can I estimate that purity?

Thank you

HI Laura,

You are looking at different types of impurity when you compare absorbance and fluorescence. With fluorescence, when you use additional kits such as a dsDNA kit when you are interested in RNA, you are looking at the contaminating nucleic acids. When you look at purity ratios using absorbance you are getting an estimate of the unwanted organic compounds such as trizol, phenol guanadine HCl etc when using the 260/230 and contaminating protein using the 260/280 ratio (or contaminating DNA in a protein sample).

Absorbance will not give you information on contaminating nucleic acids in your sample as they all absorb at 260nm. In order to get an all round picture of contamination of your sample, it is good practise to look at both the absorbance and fluorescence profiles of your sample. In broad terms, rather than using 2 x fluorescence kits you could compare e.g the fluorescence of your RNA sample to it's concentration gained by microvolume absorbance. The difference between the 2 values is a guide to how much contamination of "other" nucleic acids are in the sample There are some assumptions you have to make about fluorescence for this to hold true - such as pipetting accuracy, how appropriate the standards are to your samples etc. and that the absorbance peak at 260nm is primarily due to nucleic acid, but it generally holds well that if there is a big delta between the values then the sample is "less good"

There is a tech note on this here

https://www.denovix.com/pdf/156-Absorbance-and-Fluorescence-Quantification.pdf

or a nice infographic

https://www.denovix.com/absorbance-fluorescence-quantification-infographic/

Andy

Hi! I think that do you need read this paper.

Thank you very much!

I found a good comparison table for the same - http://www.personalizedgenes.com/nanodrop-or-qubit/

There's an even better alternative for purity or even concentration determination called the NanoPhotometer. Most specs on the market including the Nanodrop rely on surface tension because they're creating that liquid column, which needs more sample volume and your sample is completely exposed (think of evaporation). The NanoPhotometer is compressing the sample, much like a microscope slide and a cover slip creating a capillary film, which is independent from surface tension, completely enclosed (no evaporation or contamination) and allows the lowest volume on the market with 0.3ul. So measuring substances that lack surface tension such as proteins, nucleic acids and even organic solvents can be done with ease. Sometimes people tend to forget the importance of the path length used to calculate the concentration (Beer's law). The NanoPhotometer has two fixed path lengths that DO NOT change over time so you're looking at lifetime accuracy and NO MAINTENANCE because no recalibration is necessary! The technology really speaks for itself, so if you're really looking for accuracy these are things you have to factor in.

https://www.implen.de/product-page/implen-nanophotometer-np80-microvolume-cuvette-spectrophotometer/

Used any one the NanoDrop to measure cell free DNA cc prepared from blood?

it seems to me NanoDrop

http://www.personalizedgenes.com/nanodrop-or-qubit/

regards

Thank you very much @hassan nima

How qubit 3.0 quantification RNA/DNA ratio .... are there need a specific assay KIT ?

Doctor Antonio Fuertes (INC) told me that you need both of them!

Hi Francesca Ceroni

Both machines have its own aplications.

QubiT is sensible and can really read low concentration of several biomolecules with precision, also QubiT 4 can assess RNA integrity.

Nanodrop is good if you do not have to contamination at 260nm. However the new nanodrop have Acclaro sample intelligence technology which identifies presence of contaminants such as protein or guanidine and provides corrected analyte concentration value. Nanodrop can assess contamination using the ratio 260/280 and 260/230, this kind of analysis it is not possible with QubiT.

I think it is good to have both in your lab.

Best,

Jacó