Quantitation of dsDNA using Hoechst dye 33258

Many techniques of cellular and molecular biology require the ability to quantitate dsDNA in large numbers of samples at sensitivities that only require a small amount of the total sample. Although there are many different methods to quantitate DNA, most methods have disadvantages that preclude their use in some applications. Absorbance measurements at 260 nm (A260) is the most commonly used method for DNA concentration determination, but it suffers from the interfering absorbance of contaminating molecules (1). Many of these contaminants, which include nucleotides, RNA, EDTA and phenol, are commonly found in nucleic acid preparations. The fluorescent bisbenzimide (Hoechst) dyes circumvent many of these problems. Hoechst 33258 dye is relatively selective for dsDNA and in high salt does not show fluorescent enhancement in the presence of either protein or RNA. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm (2, 3). The use of Hoechst 33258 in conjunction with the BioTek Microplate Fluorescence Readers offer high specificity, as well as high sensitivity for dsDNA quantitation.

For most investigators using the dye concentrations described by Labarca and Paigen (3) are adequate, but optimal concentrations can be determined empirically. Using a Hoechst 33258 dye concentration of 1.0 mg/ml would be expected give a linear response to a wider range of DNA concentrations than lower dye concentrations. Alternatively for low levels of DNA the use of 0.1 mg/ml dye concentrations may be more appropriate, as background fluorescence is reduced. The caveat of this condition being a decrease in fluorescent signal response is resulting in a flatter dose response and saturation at lower dsDNA.

In terms of reagents this assay only requires two stock solutions. The assay buffer is composed of 2M NaCl and 50mM NaH2PO4, pH 7.4 and sterilized by autoclaving, while the stock dye solution is (1 mg/ml Hoechst dye in distilled H2O) and sterilized by filtration through a 0.22 mm filter. Both are stored at 4°C. Working solution is prepared by mixing 1 µL of dye per 1 mL of assay buffer. The fluorescence is measured in a microplate reader with a 360/40 excitation filter and a 460/40 emission filter.

This assay offers good sensitivity at a low price. As a fluorescent assay it is certainly much more sensitive than a direct absorbance at 260 nm determination, but it does require that a standard curve be used. While it is not as sensitive as a PicoGreen® assay, because the reagents, particularly the dye, are not proprietary they can be purchased at a very reasonable cost to the investigator, resulting in a cost per assay being less than $0.01. Other than the dye the assay only requires sodium phosphate for buffering and sodium chloride and the stock solutions last for extended periods of time if stored at 5°C. For the cost conscience lab that is not afraid to make their assay reagents, this is a wonderful assay and I highly recommend it. For more information regarding this DNA quantitation application using microplates check out the Hoechst application note on the BioTek website or the references listed below..

References
(1) Maniatis, T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
(2) Daxhelet, G.A., M.M. Coene, P.P. Hoet, and C.G. Cocito (1989) Analytical Biochemistry 179:401-403.
(3) Labarca, C. and K. Paigen (1980). Analytical Biochemistry 102:344-352.

Quick, Reliable Instrument Performance Verification for Low Volume Dispense Accuracy and Precision

I work in a laboratory environment where many different people use the same instruments for a diversity of microplate applications, often in the course of a single day. It only took one experiment failure for me to decide I needed a quick, reliable way to check all of my equipment before an experiment above and beyond routine instrument validation procedures of the lab.

What I came up with is not an end all solution for making every experiment successful, it doesn’t cover every fluid profile that could perform very differently than the viscosity of the test solution, nor should it be considered a replacement for regularly scheduled instrument maintenance routines, but it certainly assists in spotting potential instrument performance problems before they become expensive failures. I even use these basic dispense accuracy and precision procedures to check the performance of any manual pipettes or other appropriate accessory tools in addition to the automation before starting my laboratory work, and most manufacturers of precision tools would recommend this habit anyway.

I drew from a variety of sources to find the right mix of methods and materials, tweaking what I learned to meet my requirements of being quick and easy without losing reliability. These procedures are designed for low-volume dispense verification applicable to the 1µl, 5µl, and 10µl EL406 and MicroFlo Select cassettes, the EL406 syringe A and B dispense manifolds, and the Precision Power automated dispenser (single and multi-channel). The NanoQuot could be tested the same way using different dye dilutions for under 2µl, but this procedure isn’t geared toward that instrument. A future blog series highlighting my forays into a low volume dispense journey will refer back to these procedures, and highlight a useful application for them that can be adapted to a variety of assay/instrumentation troubleshooting scenarios.

Click here for my version of economical, dependable base line instrument verification for low volume dispensing accuracy and precision. I would be interested in knowing yours.

The Universal Utility of Green Fluorescent Protein Based Indicators

GFP was originally identified from Aequorea forskalea in the late 1960’s early 1970¹ although not characterized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP². Since that time the utility of the many variants of GFP have been critical to many research areas such as cancer research, systems biology, epigenetics, stem cell research, developmental biology and drug development and toxicity screening. In fact, the importance of GFP resulted in Martin Chalfie, Osamu Shimomura and Roger Y. Tsien sharing the 2008 Nobel Prize in Chemistry for discovery and development of GFP³. Shortly thereafter enhanced GFP (EGFP), with increased folding efficiency and stability at 37ºC, was discovered in 1995 by the lab of Ole Thastrup that opened the door for GFP usage in mammalian cells⁴. For a more comprehensive discussion of GFP see Dr. Cambell’s Scholorpedia webpage⁵.

The most popular applications of GFPs involve exploiting them for imaging, such as localization and dynamics of specific organelles and expression of chimeric or recombinant proteins. However, increased demands for higher-throughput and rapid detection have resulted in methods to exploit their use. Such assays have been adapted to a microplate format and read on fluorescent capable microplate instruments to meet the increased through-put demands. Several commercially available GFP based kits and reagents are available to assist researchers. For example GFP can be stably transfected into a model cell line to act as a gene reporter to quantify activation or inhibition at targets for pharmacological intervention⁶. Other methods focus on biochemical interactions such as cell signaling by detecting intracellular calcium mobilization⁷ or generation of hydrogen peroxide⁸. Several applications have also been developed based on fluorescent resonance energy transfer (FRET) using GFP derived proteins; the ability of one fluorescing compound to transfer, upon excitation, emission energy to excite a second molecule when in close proximity⁹. The resulting emission intensity of the second or acceptor molecule reflects the proximity of the two molecules. These applications include those where colocalization of two or more proteins can be determined below the theoretical optical resolution of current imaging technology (less than 10 nm). These types of studies are well suited to a microplate format in densities up to 1536-wells/plate. For additional information regarding applications of FRET see An Introduction to Fluorescence Resonance Energy Transfer (FRET) Technology and its Application in Bioscience on the BioTek website¹⁰.

The instrumentation itself varies in optical capability; 1) filter based optical systems allow increased light intensity, relative to monochromator based systems, for sample excitation at the filter determined wavelength and bandpass 2) monochromator based systems allow for broad wavelength and bandpass selection for both excitation and emission but exhibit decreased light intensity relative to filter based instruments. Both types of instruments have their positive and negative aspects depending on the nature of the sample and experimental conditions¹¹.

In the future we expect to see GFP used in such applications as identifying cell markers such as Oct4, Sox2 and Nanog in iPS cells in a microplate format to quantify cell induction when exposed to small molecule mimetics to endogenous transcription factors.


If this is an area you are currently working on we would like to hear from you.

As BioTek gears up our new cell culture facility over the next several months are there specific GFP or fluorescent based applications that we might help you optimize on our instruments?

References

1. Prendergast F, Mann K (1978). "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea". Biochemistry 17 (17): 3448–53.
2. Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M (1992). "Primary structure of the Aequorea victoria green-fluorescent protein". Gene 111 (2): 229–33.
3. "The Nobel Prize in Chemistry 2008". 2008-10-08. Retrieved 2009-10-14.
4. Thastrup O, Tullin S, Kongsbak Poulsen L, Bjørn S (1995). "Fluorescent Proteins". US patent. Retrieved 2009-10-14.
5. http://www.scholarpedia.org/article/Fluorescent_proteins. Retrieved 2009-10-14.
6. http://www.systembio.com/index.php?id=product_25. Retrieved 2009-10-14.
7. http://www.enzolifesciences.com/ENZ-51020/gfp-certified-fluoforte-calcium-assay-kit-for-microplates/. Retrieved 2009-10-14.
8. Belousov, V.V., Fradkov, A.F., Lukyanov, K.A., Staroverov, D.B., Shakhbazov, K.S., Terskikh, A.V., and Lukyanov, S. (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide, Nat. Methods, 3, 281-286.
9. dos Remedios, C.G. and Moens, P.D. (1995) Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor, J. Struct. Biol., 115, 175-185.
10. http://www.biotek.com/resources/articles/fluorescence-resonance-energy-transfer.html. Retrieved 2009-10-14.
11. http://americanbiotechnologylaboratory.texterity.com/abl/200608/?pg=18. Retrieved 2009-10-14.

Planet xMAP Europe 2009 Recap

"Beads can save lives." That was one of the principle messages evident at Planet xMAP Europe 2009. It was a dichotomous message referring to both the theme partner, BeadforLife and the Luminex xMAP microsphere beads. BeadforLife is an NGO that enables Ugandan women to generate cash for the bead jewelry they make from recycled paper. It helps Ugandan women afford shelter, food and the basic needs of life. The other side of the coin is the xMAP technology from Luminex and the multiplexed assays covering diverse applications across life sciences research and clinical diagnostics.


An example of xMAP technology helping to save lives was described at Planet xMAP by Dr. Christine Ginocchio, Senior Director of the Division of Microbiology at the North Shore-LIJ Health System Laboratories. The xTAG Respiratory Virus Panel (RVP) from Luminex was used to genotype influenza during a recent outbreak of swine flu (H1N1) in the Long Island, NY region. Ginocchio described that "in eight weeks time, our lab had to run almost 35,000 tests for influenza, covering 14,000 patients. Thanks to RVP, we were able to do very sensitive molecular testing ... and could tell the actual magnitude of the outbreak."


BioTek plans to assists these laboratories with their sample throughput needs with the automation of xMAP assays, using either polystyrene or magnetic microsphere beads, with our product line of microplate washers. At Planet xMAP we described in a poster the automation of the wash steps for both gene expression and protein-based multiplexed xMAP assays using the full plate washer ELx405. Over the next months we will provide new products based on the ELx50 (strip washer).


What are your throughput needs using the xMAP technology? Do you use polystyrene or magnetic microspheres?