Wavelength based dual glow reporter genes
Luminescence detection of reporter genes has been one of the key tools in a molecular biologist's toolbox for decades. With the advent of dual-reporter assays, like Promega's DLR™ assay, reporter genes became more accurate, more linear and better at accounting for expression activity and cell proliferation unrelated to the promoter of interest. Dual luciferase, however, requires the injection of two reagents, making it less amenable to high-throughput and more susceptible to artifacts due to improper mixing, detection timing or injection conditions.
New dual glow assays
Now researchers have several good options for taking advantage of dual-reporter formats with stable, long-lived luminescence reporters that emit at different wavelengths. Chroma-Glo® from Promega, Pierce-Thermo's Luciferase Dual Assay and Life Technologies' Dual-Light® are all more convenient and amenable to higher throughput detection methods. Any of these assays can be read twice as fast on a BMG LABTECH microplate reader compared to other instruments that aren't capable of simultaneous dual detection of both wavelengths.
Filters separate different luciferase signals
Firefly and Renilla luciferase enzymes emit at similar wavelengths but utilize different substrates, so they can be detected accurately at different times. Reporters that emit at different wavelengths, like Gaussia and Renilla, can be detected at the same time by detecting the different wavelengths using filters. This permits the use of glow-reactions that emit stable signals that sometimes last for hours. Cypridina Luc, for example, has an emission peak in the blue around 450nm while Red-Firefly Luc emits with a peak around 620nm. With the ability to detect luminescence emission at 450 and then at 620, the glow from one reporter doesn’t interfere with detection of the other. There are now more than a handful of luciferase constructs that emit at various wavelengths and allow researchers to design for a variety of compatibilities and conditions.
Instruments with dual emission detection
BMG LABTECH instruments are especially well-suited for the detection of dual glow reporters since they can measure two emission wavelengths simultaneously. Whereas most luminometers capable of wavelength discrimination would measure the first reporter and then return to measure the second, BMG LABTECH’s dual-emission readers can measure both at the same time, doubling the throughput. Not only does simultaneous detection halve read times, variability is dramatically reduced because fluid-movement, bubbles and temperature effects are not introduced into the calculation.
Fig. 1: Linearity and sensitivity of Chroma-Glo™ chemistry using red (CBRluc; Panel A) and green (CBG99luc; Panel B) luciferases. Cell equivalents of 300-3.000 were run on the Equator™ NS-808 and read using the PHERAstar instrument. All assays were performed with an 8µl total volume in 1536-well format.
A wider bandwidth filter gives the best performance
Monochromator-based plate readers typically do not work well for dual glow luciferase reporters. Most monochromators have a fixed, narrow bandpass of 20nm or less. Luciferase emission is generally quite broad. In the case of green-Renilla, from 450- to 650nm - BMG LABTECH designs filters specifically for dual glow reporters that let light in broadly over the entire peak, allowing as much as 20x more light to be detected. More light means lower measurement times, which results in a synergy with our plate readers.
Dual emission detection together with faster filter-based wavelength selection means that read times may be four times faster on a BMG LABTECH reader than most other luminometers.
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