Qubit fluorometer
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The Qubit fluorometer is a laboratory instrument developed and distributed by Invitrogen, which is now a part of Thermo Fisher. It is used for the quantitation of DNA, RNA and protein.[1][2][3][4]
Method
The Qubit fluorometer uses fluorescent dyes to determine the concentration of either nucleic acids or proteins in a sample. Specialized fluorescent dyes bind specifically to the substances of interest. A spectrophotometer is used in this method to measure the natural absorbance of light at 260 nm (for DNA and RNA) or 280 nm (for proteins).[5][6][7][8]
Fluorescent dyes
The Qubit assays (formerly known as Quant-iT) were previously developed and manufactured by Molecular Probes (now part of Life Technologies). Each dye is specialized for one type of molecule (DNA, RNA or protein). These dyes exhibit extremely low fluorescence until bound to their target molecule. Upon binding to DNA, the dye molecules assume a more rigid shape and increase in fluorescence by several orders of magnitude, most likely due to intercalation between the bases.[9][10]
The Qubit fluorometer picks up the fluorescence signal from the sample and converts it into a concentration measurement by referring to 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 biomolecules and concentrations ("ds" stands for double-stranded, and "ss" for single-stranded DNA):
| 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 |
Versions
The second generation, the Qubit 2.0 Fluorometer, was released in 2010, and the 3rd generation as Qubit 3.0 in 2014. The newest version is the 4th generation Qubit 4, which was launched in 2017.
References
- ^ Acar E, et al. (2009). "Optimization and validation studies of the MentypeR Argus X-8 kit for paternity cases". Forensic Sci Int Genet Suppl. 2: 47–48. doi:10.1016/j.fsigss.2009.08.189.
- ^ Bakos J, et al. (2009). "Enriched environment influences hormonal status and hippocampal brain derived neurotrophic factor in a sex dependent manner". Neuroscience. 164 (2): 788–797. doi:10.1016/j.neuroscience.2009.08.054. PMID 19723563. S2CID 23809910.
- ^ Halaihel N, et al. (2009). "A new real time PCR-based assay for diagnosing Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) and comparison with other techniques". J Microbiol Methods. 76 (1): 75–80. doi:10.1016/j.mimet.2008.09.014. PMID 18938198.
- ^ Hamza IA, et al. (2009). "Detection and quantification of human bocavirus in riverwater". J Gen Virol. 90 (Pt 11): 2634–2637. doi:10.1099/vir.0.013557-0. PMID 19656966.
- ^ Manchester, K.L. (1996). "Use of UV methods for the measurement of protein and nucleic acid concentrations". BioTechniques. 20 (6): 968–970. doi:10.2144/96206bm05. PMID 8780864.
- ^ Glasel, J.A. (1995). "Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios". BioTechniques. 18 (1): 62–63. PMID 7702855.
- ^ Huberman, J.A. (1995). "Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm". BioTechniques. 18 (4): 636. PMID 7598897.
- ^ Manchester, K.L. (1995). "Value of A260/A280 ratios for measurement of purity of nucleic acids". BioTechniques. 19 (2): 208–210. PMID 8527139.
- ^ McKnight, R.E., Gleason, A.B., Keyes, J.A., Sahabi, S. (2006). "Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay". Bioorganic & Medicinal Chemistry Letters. 17 (4): 1013–1017. doi:10.1016/j.bmcl.2006.11.038. PMID 17157016.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Schweitzer, C., Scaiano, J.C. (2003). "Selective binding and local photophysics of the fluorescent cyanine dye PicoGreen in double-stranded and single-stranded DNA". Physical Chemistry Chemical Physics. 5 (21): 4911–4917. Bibcode:2003PCCP....5.4911S. doi:10.1039/b305921a.
{{cite journal}}: CS1 maint: multiple names: authors list (link)