A review of the Qubit fluorometer for quantifying DNA, RNA, and proteinsArticle created: Feb 18, 2008
Last update: Jun 13, 2008
Article by: Jeremiah Faith
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.
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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
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.
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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).
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