 | ||
The Qubit fluorometer is a small instrument used for quantification of DNA, RNA, and protein and used in many different applications. The second generation of this instrument, the Qubit 2.0 Fluorometer, was released Nov. 15, 2010. The newest model, the Qubit 3.0, was released in 2014.
The Qubit fluorometer uses fluorescent dyes to determine the concentration of nucleic acids and proteins in a sample. The UV-absorbance method uses a spectrophotometer to measure the natural absorbance of light at 260 nm (for DNA and RNA) or 280 nm (for proteins). The more DNA, RNA or protein in the sample, the more light is absorbed. The absorbance is a natural property of DNA, RNA, free nucleotides, proteins and some amino acids and many other compounds as well. Because so many molecules absorb light at 260 nm, this measurement is subject to inaccuracy due to potential contamination of the sample with these other molecules. In addition, using the absorbance method, it is not possible to distinguish between DNA, RNA, protein or free nucleotides or amino acids in the sample, leading to potentially highly inaccurate measurements.
The Qubit assays (previously known as Quant-iT) used with both the original Qubit fluorometer and the newer 2.0 fluorometer were developed and manufactured by the previous Molecular Probes (now a part of Life Technologies). Each dye is specific for one type of molecule: DNA, RNA or protein. These dyes have extremely low fluorescence until they bind to their targets (DNA, RNA or protein). Upon binding, they become intensely fluorescent. The difference in fluorescence between bound and unbound dye is several orders of magnitude. For example, the Qubit DNA dye used for the high sensitivity assay has extremely low fluorescence until it binds to DNA. Upon binding to DNA, probably by intercalation between the bases, it assumes a more rigid shape and becomes intensely fluorescent. Once added to a solution of DNA, the Qubit DNA dye binds to the DNA within seconds and reaches equilibrium in less than two minutes.
At a specific amount of the dye, the amount of fluorescence signal from this mixture is directly proportional to the concentration of DNA in the solution. The Qubit fluorometer can pick up this fluorescence signal and convert it into a DNA concentration measurement using DNA standards of known concentration. The Qubit fluorometer uses DNA standards to derive the relationship between DNA concentration and fluorescence. It then uses this relationship to calculate the concentration of a sample, based on its fluorescence when mixed with the Qubit dye.
Other fluorometers can also measure the fluorescence from the Qubit dyes and can be used for DNA, RNA and protein quantification in the same way. However, all other fluorometers require the user to use several DNA standards and plot the concentration versus the absorbance on a graph. The data must then be fitted to a line and finally the sample concentration calculated from the equation of the line. Although this is a simple calculation for any scientist, the Qubit fluorometer does this calculation for the user, making it faster and easier, in addition to being less expensive than a typical fluorometer.
The entire Qubit quantitation system includes the following dyes that are specific for different bio-molecules.
The advantage of using these specific dyes is that the researcher can tell precisely how much of each bio-molecule (DNA, RNA or protein) is in a specific sample, even in the presence of other bio-molecules. For example, one of the DNA quantification kits may be used to measure DNA concentration, one of the RNA kits to measure RNA concentration, and the protein kit to measure protein concentration of the same stock sample. Together, they give the concentrations of DNA, RNA, and protein in a sample.