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A review of the Qubit fluorometer for quantifying DNA, RNA, and proteins

Article created: Feb 18, 2008
Last update: Jun 13, 2008
Article by: Jeremiah Faith


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The Qubit fluorometer is a device from Invitrogen for quantifying proteins, RNA, or DNA. It uses various Quant-iT assays, which contain sensitive dyes that fluoresce in proportion to the amount of protein, RNA, or DNA respectively. The dyes themselves can be used with any fluorescent plate reader, but the Qubit device is so easy to use and not too costly that I’d buy another if my current one broken rather than run the assays in our lab’s plate reader.

How the Qubit works

The Qubit is relatively cheap when compared with other fluorometers, because it uses colored LEDs as light sources. The device contains a Blue LED and a Red LED and detects the fluorescence with a photodiode. Each of the Quant-iT assay kits comes with a buffer, an assay specific dye, and two standards (high and low). Two standards are prepared with a fixed amount of nucleic acid or protein (depending on the kit; and provided with the kit) combined with buffer and dye. The samples to be quantified are prepared similarly using 1-20 ul of each sample combined with buffer and dye. All standards and samples are run in clear 500 ul tubes that are available from Invitrogen (and not too expensive).

To quantify your samples, the Qubit will first ask for your low concentration standard and then for your high concentration standard. It uses these two measurements to fit a standard curve. The parameters from this standard curve are then used to estimate the quantity of your samples using the fluorescence of your sample and linear regression.

To see how the Qubit works in action, Invitrogen has created a Qubit Virtual Demo.

Positive features of the Qubit and Quant-iT assays



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Since each Quant-iT assay requires a particular dye and tube there is a fixed cost per sample; however this fixed cost is quite low (currently less than a dollar per sample). One extremely useful aspect of using this dye-based quantification is that the dyes are quite specific. For example if you extract RNA from cells, there is often some residual genomic DNA in your sample. The genomic DNA contributes very little fluorescence to your overall signal if you use the RNA specific Qubit dye, so you can quantify only the RNA (which presumably is what you’re interested in); similarly, if you run a 1st strand cDNA synthesis, you likely have RNA in your sample, but you can estimate the amount of DNA resulting from your cDNA synthesis using a DNA specific Qubit dye. This specificity is a big advantage over standard spectrometer based quantification of nucleic acids which can only lump the total amount of RNA and DNA into one quantity.

The dyes, particularly the dsDNA HS (HS = high sensitivity) dye, are very sensitive and allow quantification of DNA at concentrations far below the limits of a spectrophotometer (from 10 pg/ul to 100 ng/ul, whereas I find the lower limit for the Nanodrop spectrophotometer is around 20 ng/ul). The RNA dye isn’t quite as sensitive as the dsDNA HS dye, but still enables quantification of RNA far below the limits of a Nanodrop spectrophotometer (from 250 pg/ul to 100 ng/ul, again the practical limit for the Nanodrop is around 20 ng/ul).

The Quant-iT dyes are also quite insensitive to the presence of salts and other contaminants. I was never really able to accurately measure the amount of DNA remaining from my gel purifications with a spectrophotometer, but the Qubit can measure them just fine. With the Qubit, I’ve even been able to quantify extremely low concentration DNA samples like ChIP DNA for the first time.

Negative features of the Qubit and Quant-iT assays

Setting up a Qubit based quantification is slightly slower than using a spectrophotometer like the Nanodrop, because you have to prepare the dye and buffer for each sample, you have to prepare two standards for each set of samples, and you have to place each sample and standard in its own tube for quantification. In addition, the Quant-iT dyes come as a 200x solution in DMSO which is stored frozen at 4C (DMSO melting point is 18.5C) and takes quite a while to melt at room temperature; so it takes a little extra planning to make sure the dye is ready for your experiment. Because of the extra time and cost for running the Qubit when compared with the Nanodrop spectrophotometer, I use the Nanodrop for pure samples (i.e. containing only RNA or DNA and not a mix of the two) above 30 ng/ul.

Another negative aspect of the Qubit is that it requires a lot of sample if the sample has an extremely low concentration. You prepare the quantification reaction with 1-20 ul of sample. For high concentration samples 1-2 ul is fine, but for extremely low concentration samples it is necessary to use 10-20 ul to have enough nucleic acid or protein to be detected by the fluorometer. Unfortunately, samples are typically extremely low concentrated when you don’t have much of them to begin with, so to quantify them with the Qubit you often need to use almost the whole sample. For example, to quantify my ChIP DNA I must quantify 10 ul of a 30 ul sample to estimate the total amount of DNA in the sample. But at least it is possible to quantify it.

My least favorite aspect of the Qubit is that you need to have at least a crude guess of your sample concentration to know which dye to use and to know what amount of sample to use in the quantification . otherwise you’ll go outside the bounds of the machine. For example, if 1 ul of a high concentrated sample contains more DNA per microliter than the high standard for calibration, you’ll get an out-of-range error. When your sample is above the range, you can either dilute it or quantify the sample with a spectrophotometer.

Update

It turns out my earlier statement that you need to thaw the dye each time you use it was not correct. I received the following info in an email from Jill Hendrickson, the Qubit Project Manager at Invitrogen: >”I also wanted you to know that we did design the kits so that you can store the dye and buffer at room temperature. That’s because we knew it would be a pain to thaw the dye out. You just need to store the dye in the dark, like in a drawer.” After looking at my qubit reagent tubes a bit, I did see that although the large container the kit comes in says 4C the individual tubes have their own storage condition labels, and the dye label says to store at less than or equal to 25C (room temperature). This prevents having to plan your experiments around the melting time of the dye and makes the Qubit much easier to use.

Conclusions



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The most important aspect of wetlab experiments in general and novel experimental technique development in particular is knowing what you have in your sample and how much you have of it. Since purchasing the Qubit, I’ve found multiple occasions where I previously had to guesstimate the amount of RNA or DNA in my sample but with the Qubit I could accurately and quickly quantify my sample. For the bulk of my wetlab work which involves high concentration RNA or DNA samples that are relatively pure, I prefer to use the Nanodrop spectrophotometer which is faster to set up and has no per-sample reagent cost. But for those cases where a sample isn’t pure enough or concentrated enough, the Qubit has become an essential part of my molecular biology toolkit.