PicoGreen

PicoGreen is a highly sensitive fluorescent probe developed by Invitrogen, specifically designed to detect double-stranded DNA (dsDNA, Dragan, A.I., et al., 2010). Upon binding to dsDNA, PicoGreen undergoes a conformational change that results in the formation of a fluorescent complex, emitting light upon excitation. PicoGreen reagents are commonly used in molecular biology applications such as quantitation of dsDNA. The broad detection range of the dye (25 pg/mL to 1,000 ng/mL) allows for precise quantification across four orders of magnitude (Demeke & Jenkins, 2009). To ensure accurate quantification, stock solutions should be properly prepared according to the manufacturer's PicoGreen protocol.

Several key properties contribute to the use of PicoGreen in molecular biology applications. One notable characteristic of PicoGreen is its high molar extinction coefficient, which is approximately 70,000 cm-1 M-1 (Singer et al., 1997). This high extinction coefficient indicates that the dye is efficient at absorbing light at its maximal absorbance wavelength, allowing for a bright fluorescence signal to be emitted upon excitation by the appropriate light source; PicoGreen has an excitation maximum at 502 nm and an emission peak at 520 nm. In addition to a high extinction coefficient, the dye also possesses a complex chemical structure designed to optimize its binding and fluorescence properties. Its structure includes a quinolinium core, specifically 2-(n-bis-(3-dimethylaminopropyl)-amino)-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenyl-quinolinium. Because of these properties, PicoGreen assays, such as Quant-iT kits, can accurately quantify dsDNA. The assay principle for these PicoGreen based kits relies on measurements of fluorescence increase, which is directly proportional to the amount of dsDNA present in the sample.

The main advantages of using PicoGreen assays are that they are reliable, straightforward, and fast methods for quantifying DNA, typically taking less than 30 minutes (Zhang, S. B., et al., 2015). It also works across a broad range of DNA concentrations and only requires a basic fluorescence reader. Furthermore, DNA measurements obtained from plasma using the PicoGreen assay show strong correlation with real-time qPCR results. The methodology is also simple and easy to learn, making it accessible for most users. Moreover, the limit of detection for the PicoGreen assay is below 1.0 ng of double-stranded DNA per sample, independent of the detection method or the complexity of the sample. In practical use, the assay can accurately measure approximately 250 ng of genomic DNA per sample and around 1.0 ng of a small PCR product in a microtiter format (Ahn, 1996). One potential point to note, however, is that PicoGreen can also bind to double-stranded RNA (dsRNA), including miRNA mimics and siRNA when these RNA molecules are in solution (Ban & Kim, 2024).

While PicoGreen is widely used, there are several alternatives. A direct alternative is Helixyte, which offers the same dsDNA quantification performance, but at a lower price point. Another alternative is CytoQuant GR. The difference between PicoGreen and CytoQuant is that the former is used to quantify dsDNA while the latter is used more for cell proliferation applications. Additionally, PicoGreen and CyQuant differ in their binding specificity and sensitivity, with the former binding specifically dsDNA while the latter binds nucleic acids, including RNA, but with a preference for DNA (Quent, V. M. C., et al., 2010). Looking at assay kits, the Quant-iT PicoGreen kits have an alternative in the form of Qubit dsDNA assays. The difference between PicoGreen vs Qubit in this case is that Qubit assay kits tend to perform better when quantifying dsDNA at the lower end of the detection range. A fourth and final alternative to PicoGreen is the NanoDrop. The difference between PicoGreen and NanoDrop is that the former is fluorescence based, while the latter is absorbance based. NanoDrop is a spectrophotometer that utilizes UV absorbance to measure nucleic acid concentration, specifically by reading at the 280 nm wavelength. The disadvantage of the NanoDrop, compared to PicoGreen, is that it cannot differentiate between DNA, RNA and free nucleotides. In contrast, PicoGreen is designed to specifically quantify dsDNA with much greater sensitivity. (Versmessen et al., 2024).

References

Ahn, S. "PicoGreen Quantitation of DNA: Effective Evaluation of Samples Pre- or Post-PCR." Nucleic Acids Research, vol. 24, no. 13, 1 July 1996, pp. 2623-2625, https://doi.org/10.1093/nar/24.13.2623.
Ban, Eunmi, and Aeri Kim. "PicoGreen Assay for Nucleic Acid Quantification - Applications, Challenges, and Solutions." Analytical Biochemistry, vol. 692, 1 May 2024, pp. 115577-115577, https://doi.org/10.1016/j.ab.2024.115577.
Demeke, Tigst, and G. Ronald Jenkins. "Influence of DNA Extraction Methods, PCR Inhibitors and Quantification Methods on Real-Time PCR Assay of Biotechnology-Derived Traits." Analytical and Bioanalytical Chemistry, vol. 396, no. 6, 30 Sept. 2009, pp. 1977-1990, https://doi.org/10.1007/s00216-009-3150-9.
Dragan, A.I., et al. "Characterization of PicoGreen Interaction with DsDNA and the Origin of Its Fluorescence Enhancement upon Binding." Biophysical Journal, vol. 99, no. 9, 3 Nov. 2010, pp. 3010-3019, www.ncbi.nlm.nih.gov/pmc/articles/PMC2965993/, https://doi.org/10.1016/j.bpj.2010.09.012.
Quent, Verena M.C., et al. "Discrepancies between Metabolic Activity and DNA Content as Tool to Assess Cell Proliferation in Cancer Research." Journal of Cellular and Molecular Medicine, vol. 14, no. 4, 15 Jan. 2010, pp. 1003-1013, https://doi.org/10.1111/j.1582-4934.2010.01013.x.
Singer, Victoria L., et al. "Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation." Analytical Biochemistry, vol. 249, no. 2, July 1997, pp. 228-238, https://doi.org/10.1006/abio.1997.2177.
Versmessen, Nick, et al. "Comparison of DeNovix, NanoDrop and Qubit for DNA Quantification and Impurity Detection of Bacterial DNA Extracts." PLoS ONE, vol. 19, no. 6, 17 June 2024, pp. e0305650-e0305650, https://doi.org/10.1371/journal.pone.0305650.
Zhang, Steven B., et al. "PicoGreen Assay of Circular DNA for Radiation Biodosimetry."" Radiation Research, vol. 183, no. 2, Feb. 2015, pp. 188-195, https://doi.org/10.1667/rr13556.1.

Original created on December 11, 2024, last updated on December 11, 2024
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