Qubit fluorometer
The Qubit fluorometer is a lab instrument developed and distributed by Invitrogen that, among other applications, is used for the quantification of DNA, RNA, and protein.
Principle
The Qubit fluorometer uses fluorescent dyes to determine the concentration of either nucleic acids or proteins in a sample. The other common method of measuring the concentration of nucleic acids and protein is the UV-absorbance method, which uses a spectrophotometer to measure the natural absorbance of light at 260 nm or 280 nm. 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 and is unable to distinguish between DNA, RNA, protein or free nucleotides or amino acids in the sample. On the other hand, Qubit system is supplied with fluorescent dyes that bind specifically to analytes of interest such as double-stranded DNA, single-stranded DNA, RNA, miRNA or protein providing more accurate quantification.Fluorescent dyes
The Qubit assays were developed and manufactured by the previous Molecular Probes. Each dye is specific for one type of molecule. They have extremely low fluorescence until bound to their target molecule. The difference in fluorescence between bound and unbound dye is several orders of magnitude. Upon binding to DNA, probably by intercalation between the bases, the dye molecules assume a more rigid shape and become 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 concentration of the dye, the intensity of the fluorescence signal from this mixture is directly proportional to the concentration of DNA in the solution, even in the presence of other bio-molecules. The Qubit fluorometer picks this fluorescence signal up and converts it into a DNA concentration measurement by referring to DNA probes of known concentration. It then uses this relationship to calculate the concentration of a sample.
The Qubit quantitation system includes the following dyes that are specific for different bio-molecules and concentrations :
| Reagent/Assay | Assay range | Sample starting concentration range |
| Qubit dsDNA HS Assay | 0.2–100 ng | 10 pg/μl–100 ng/μl |
| Qubit dsDNA BR Assay | 2–1,000 ng | 100 pg/μl–1 μg/μl |
| Qubit ssDNA Assay | 1-200 ng | 50 pg/µL-200 ng/µL |
| Qubit RNA Assay | 5–100 ng | 250 pg/μl–100 ng/μl |
| Qubit RNA BR Assay | 20-1,000 ng | 1 ng/µ-1 µg/µL |
| Qubit Protein Assay* | 0.25–5 μg | 12.5 μg/ml–5 mg/ml |