Techne PrimeQ Filters

PrimeQHelp@bibby

-

scientific.com

| www.bibby

-

scientific.com

[phone]

1

Application Note A08-002A

FC04 580 615 ROX™,

TEXAS RED®, Cy®3.5

FAM™

495 520

PrimeQ Filters

Filter Selection

When performing experiments requiring detection of more than one fluorophore, it is
highly desirable for minimal overlap in signal from each dye. Whilst the choice of
fluorophore used is important, the design of the optics for sample excitation and emission
detection is crucial to obtain a sensitive and specific assay. The most specific method of
exciting and detecting multiple dyes is to use a series of tailored excitation and emission
filters. These ensure that only light of the correct wavelength reaches the dye to be
excited and only emitted light of the correct wavelength reaches the detector.

PrimeQ filter cartridge design incorporates both excitation and emission filters together with a dichroic mirror for
accurate fluorophore excitation and emission detection. The filter cartridges available for PrimeQ cover the
spectrum of popular fluorescent dyes (Table 1).

Filter cartridge Excitation λ (nm) Emission λ (nm) Suitable dyes

FC01 460 500 FAM™ multiplex, SYBR® Green I, Fluorescein, EvaGreen®
FC02 485 520 FAM™, SYBR® Green I, Fluorescein, EvaGreen®
FC03 530 560 HEX™, TET™, JOE™, VIC®, Yakima Yellow®

FC05 640 685 Cy®5, Quasar® 670

Table 1: PrimeQ filter cartridges for the most commonly used dyes.

FC02, FC03, FC04 and FC05 are supplied as standard with PrimeQ and are optimised for use with FAM™, HEX™,
ROX™ and Cy®5. For multiplexing FAM™ with alternative dyes which are detected with the FC03 filter, we
recommend the use of FC01 which has lower excitation and emission wavelengths and a slightly wider bandwidth.

Crosstalk

Crosstalk can occur when the emission of one fluorophore is detected through the filter combination of another
fluorophore. Using specifically designed filters minimises or eliminates this effect and removes the requirement for
complex colour compensation algorithms. To demonstrate the specificity of PrimeQ filters, a number of
oligonucleotides were synthesised and labelled with various fluorescent dyes and HPLC purified (Table 2). Each
dye-labelled oligo was diluted to the same final concentration and read with each of the five filters.

Fluorophore Excitation λ (nm) Emission λ (nm)

JOE™ 529 555
TET™ 521 536
Yakima Yellow® 531 549
HEX™ 535 556
Cal Fluor® Orange 560 538 559
Cy®3 555 565
TAMRA™ 557 583
ROX™ 586 610
Cy®3.5 581 596
TEXAS RED® 589 615
Cal Fluor® Red 635 618 637
Cy®5 646 662

Table 2: Fluorophores tested with the PrimeQ filters. The excitation and emission wavelengths are given.

PrimeQHelp@bibby

-

scientific.com

|www.bibby

-

scientific.com

[phone]

2

A08-002A: PrimeQ Filters

Figure 1 demonstrates the range and specificity of PrimeQ filters. The overlap between certain filters is clear. This
illustrates that correct pairing of filter with fluorophore avoids the issue of crosstalk.

Figure 1: Fluorescence of various fluorophores read with

each of the PrimeQ filters.

Conclusions

Using a combination of excitation and emission filters to eliminate crosstalk between dyes provides confidence in
knowing that a signal has been generated by a specific probe without contribution from any other possible
reactions in the same well. In addition, the fluorescent data does not have to be manipulated in any way in order
to subtract any contribution to the signal by other dyes. This means that the results obtained are exactly what the
detector sees i.e. the raw data, whether performing multiplex or single dye experiments.

The fluorescent dyes FAM™, HEX™, ROX™ and Cy®5 show no cross talk when read using PrimeQ filters FC02, FC03,
FC04 and FC05, therefore we would recommend using a combination of these four dyes when designing probes for
a multiplex assay. Other available dyes are also suitable, as shown in Table 1. However if the excitation or emission
maxima of the chosen dye are shifted significantly to shorter or longer wavelengths then some cross talk may
occur. Use of the FC01 filter may be advantageous in these applications where FC03 is used to read probes labelled
with TET™, JOE™, VIC® or Yakima Yellow®. An advantage of the PrimeQ filter cartridge system is that any custom
filter cartridge can be designed, thus allowing flexibility and the capability to use many current and future dyes.

Trademarks

Cy3, Cy3.5, Cy5, are registered trademarks of GE Healthcare.
Yakima Yellow® is a registered trademark of Epoch Biosciences, Inc.
Cal Fluor® and Quasar® are registered trademarks of Biosearch Technologies, Inc.
FAM™, HEX™, JOE™, TAMRA™, ROX™ and TET™ are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
VIC® is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
SYBR® and TEXAS RED® are registered trademarks of Life Technologies Corporation.
EvaGreen® is a registered trademark of BIOTIUM, INC.