Technical Note. Roche Applied Science. No. LC 19/2004. Color Compensation

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1 Roche Applied Science Technical Note No. LC 19/2004 Purpose of this Note Color The LightCycler System is able to simultaneously detect and analyze more than one color in each capillary. Due to overlap of the emission spectra of the dyes, one channel picks up signals (crosstalk) from a dye measured by another channel. This bleed-over of fluorescence signal can result in data that cannot be interpreted. To correct the bleed-over, color compensation can be applied either during an experimental run or before data analysis. When color compensation is activated, LightCycler algorithms use the data from a so-called ccc file (LightCycler SW 3.5) or ccc object (LightCycler SW 4.0) to compensate for the fluorescence crosstalk. This Technical Note will show you how to create color compensation for dual color analyses with LC Red 640 and LC Red 705. In addition, it describes the color compensation procedure for multiplex assays. Note: Color compensation for multiplex assays can only be done on the LightCycler 2.0 Instrument. Table of Contents Basic Information about Color 2 Generating Color 4-6 Color for Dual Color Applications that Use LightCycler Red 640 and LightCycler Red 705 Creating Color Files with SW 3.5 Creating Color Objects with SW 4.0 Color for Customized Applications 7 Applications Using Customized Dual Color Hybridization Probes 8 Multicolor Hybridization Probe Applications 8-10 Application Example 11 Note: Methods of color compensation for Dual Color Hydrolysis Assays (conventionally called TaqMan Assays) will be found in Technical Note No. LC 21, Hydrolysis Probe ( TaqMan ) Assays with the LightCycler System.

2 1. Basic Information about Color Dyes and Detection Channels Spectral Overlap The LightCycler System can detect two or more different sequences in one sample, e.g., both a target and an internal control. Such assays require two specific sets of hybridization probes, each labeled with different FRET (Fluorescence Resonance Energy Transfer) acceptor dyes. Example: For dual color detection, one of the two hybridization probe sets uses LightCycler Red 640 as acceptor dye; the other uses LightCycler Red 705. Both acceptor dyes are excited by the donor dye fluorescein. These red dyes have different emission spectra, which allows the LightCycler Instrument to detect the distinctive signal from each set in different fluorimeter channels. The LightCycler 2.0 Instrument has additional detection channels that make it possible to perform multicolor (e.g. multiplex hybridization probe) assays with four different acceptor dyes: LightCycler Red 610, LightCycler Red 640, LightCycler Red 670 and LightCycler Red 705. Note: For more details of hybridization probe chemistry please read Technical Note No. LC 18/2004, Assay Formats for Use in Real-Time PCR. Although the optical filters of each detection channel are optimized for different emission maxima, all fluorescent dyes currently available have emission spectra with long tails, leading to a spectral overlap. As depicted in Figure 1, the emission spectrum of the donor dye (fluorescein) slightly overlaps the emission spectrum of LightCycler Red 640, which, in turn, overlaps the LightCycler Red 705 emission spectrum. Figure 1: Overlap of emission spectra from fluorescent dyes used in the LightCycler System. The gray vertical bars in Figure 1 show the width of the detection windows in the 530 nm, 640 nm and 705 nm channels of the LightCycler. The hatched areas below these bars represent the bleed-over (overlap) of the fluorescein and LightCycler Red 640 signals into other channels. There is especially significant overlap of the 640 nm signal into the 705 nm channel. Calibration with Color 2 You can easily correct the spectral overlap described above by calibrating the instrument with the LightCycler Color Set (Cat. No , for dual color applications that use LightCycler-Red 640 and LightCycler-Red 705) or by customized calibration (for applications that use the additional channels of the LightCycler 2.0 Instrument). During a calibration run, the LightCycler Instrument measures the fluorescence of each dye in all channels and generates an instrument-specific color-compensation file (or object). Later, the software automatically uses this so-called ccc file or ccc object to reassign the fluorescence in each channel to the appropriate dye. The net result is detection of only one dye signal in each channel. Note: Color compensation is only necessary when you run an experiment with two or more different dyes in one capillary. Color compensation is not required when the experiment uses only a single dye (e.g., SYBR Green I or LightCycler Red 640).

3 1. Basic Information about Color continued Using Factors Influencing the Relative Crosstalk Compensating for Temperature Compensating for Optical System Minimizing Effects of Coupling Chemistry Color compensation files or objects are required for accurate data analysis of dual color/multicolor assays. They may also be used to correct on-line data display during the LightCycler Instrument run. The following factors strongly influence the degree of crosstalk: temperature properties of the optical system coupling chemistry of the fluorescence dyes On the other hand, neither the final reaction volume nor any of the components of LightCycler reagents and kits that are optimized for PCR and RT-PCR significantly influence crosstalk. Note: For multicolor applications with more than two dyes and applications with adjacent fluorescent dyes (e.g. LightCycler Red 640 combined with LightCycler Red 670), the oligonucleotide sequence of the probes can influence (and usually increase) crosstalk. Thus, we recommend customized, assay-specific color compensation runs for such assays. The intensity and the spectrum of emissions from a fluorescent dye are strongly temperature dependent. Thus the temperature chosen for data acquisition (fluorescent measurement) during a PCR run significantly influences the crosstalk. This is particularly important during analysis of data measured at different temperatures (e.g., during a melting curve analysis). To provide crosstalk compensation at all temperatures, a color compensation calibration run must include a temperature gradient step (40 C 95 C) in which data from all fluorescent dyes are continuously measured in all channels. Optical systems (containing e.g. several optical filters, lenses and dichroic mirrors) will always show minor variations from instrument to instrument. This is a property of all optical systems, not just the optics in the LightCycler Instrument. If you look at raw data rather than calculated data, you can see these minor variations. Moreover, these hardware variations influence crosstalk values. Therefore, you must generate and store an instrument-specific color compensation file before starting multicolor applications on that LightCycler Instrument. The chemistry used to couple fluorescent dyes to probes can influence crosstalk. To minimize these effects, use specific labeling reagents that are available from Roche Applied Science, e.g. LightCycler Fluorescein CPG. Alternatively, if you let licensed oligonucleotide synthesis companies make your labeled probes, they will use these same high quality labeling reagents. Note: For details, see Biochemica No. 1/2001, p

4 2. Generating Color Different Possibilities for Color Color Run You can create an instrument-specific color compensation file (or object) for: Dual color applications that use LightCycler Red 640 and LightCycler Red 705 Note: Prepare this ccc file/object with the ready-to-use LightCycler Color Set. The LightCycler Software provides pre-defined protocols for this color compensation run. For details, see sections and Customized dual color applications that use HybProbe probes labeled with other dye combinations Customized multicolor applications (e.g., multiplex PCR or multicolor detection) Note: Applications involving dyes other than Red 640 and Red 705 can only be used in the LightCycler 2.0 Instrument. The temperature profile used in a color compensation protocol always includes a Heating Step, a Cycling Step, a Temperature Gradient Step and a Cooling Step. The cycling step mimics a typical PCR, including data acquisition. However, the data required for color compensation are taken from the temperature gradient step. In this step, after a brief denaturation (95 C), the protocol slowly increases the temperature, in increments of 0.2 /sec, from 40 C to 95 C while continuously acquiring data (see Fig. 2). After the calibration run, the LightCycler Software saves the data generated as a normal experimental file. For these data to be used for color compensation, you must first convert the data into a ccc file/object and save it again. Figure 2: Temperature profile that includes a temperature gradient step 4

5 2.1 Color for Dual Color Applications that Use LightCycler Red 640 and LightCycler Red 705 Overview: Dual Color with the Color Set The following sections describe how to prepare a color compensation file for a dual color experiment (using LC 640 and LC 705) on two different versions of the LightCycler Software, SW 3.5 and SW 4.0. You only need to use the LightCycler Color Set once to generate a color compensation file or color compensation object that can be used for all dual color (LC 640/LC 705) experiments on that instrument. The only times you need to generate a new ccc file/object are when you update the software or after the optical system has been repaired. The set contains four vials of ready-to-use solutions: Vial 1: standard buffer Vial 2-4: standard oligonucleotides labeled with either fluorescein (3 end; Vial 2), LightCycler Red 640 (5 end; Vial 3) or LightCycler Red 705 (5 end; Vial 4) Creating Color Files with SW 3.5 Running a Color Experiment Creating Color Files with SW 3.5 For details on running the color compensation experiment, see the pack insert of the LightCycler Color Set (new Cat. No ) and the LightCycler Operator s Manual. To perform the color compensation calibration experiment, use the calibration.exp file, which you can find in the Demo Protocol folder under Demo Data. It contains all the necessary sample information. After the calibration run, the LightCycler software automatically displays the LightCycler Data Analysis module. To convert the acquired data: Use the Select a Program function to select the melting curve segment (segment 3) of the temperature profile. (On the display, the software will place green cursors around the temperature gradient step.) Then select Calibration from the pull-down Color menu. Save the Color data by naming the calibration file and placing it in the proper folder. (The software automatically adds the.ccc extension to the file.) 5

6 2.1.1 Creating Color Files with SW 3.5 continued Applying Color Files You can activate the stored color compensation file (*.ccc file) at several points in the LightCycler Protocol to provide color compensation for raw data: On-line color compensation You can load the *.ccc file during a run on the LightCycler Instrument to correct the on-line display of data. Just click on Choose CCC File and select the appropriate file. Color compensation for data analysis You can load and activate the *.ccc file to compensate for crosstalk between channel 2 and channel 3 during dual color analysis. Before starting this analysis, just select Load Calibration Data on the Color menu, then select the appropriate file Creating Color Objects with SW 4.0 Running a Color Experiment Creating Color Objects with SW 4.0 For details on running the color compensation experiment, see the pack insert of the LightCycler Color Set (new Cat. No ) and the LightCycler Operator s Manual. To create the color compensation object, use the macro for automatic execution of the calibration, which is included in the Roche folder under Templates and Macros / Experiment Macros / Demo Macros / Color Set (Cat No ). Follow the instructions provided by the Experiment Kit Wizard. After the calibration run, select Color from the Analysis menu and save the data as a ccc object: Click the Save CC Object button. Place the object in the Special Data\CCC folder under your user name. 6

7 2.1.2 Creating Color Objects with SW 4.0 continued Applying Color Objects You can apply the stored color compensation object to all dual color HybProbe experiments which use the same dye combination (Red 640/Red 705) by doing the following: Click on Color in either the Run or the Analysis module, then choose the appropriate object under Select Color. When a dialog box opens and displays all compensated channels, click OK. Note: Do not deselect the pre-defined channels. Uncompensated channels in an experimental data set are not displayed during analysis. Result: The analysis graphs will now be redrawn using the compensated data. Notice that the Color menu label now says On. 2.2 Color for Customized Applications Introduction LightCycler 2.0 Instrument Properties You will need to develop a customized color compensation object if you want to perform an assay with: a HybProbe pair that uses different fluorophores (e.g. LC Red 610/LC Red 670) than those in the LightCycler Dual Color Set, or more than two colors of fluorophore. (Most Roche parameter-specific assay kits that use multicolor analysis include color compensation reagents that can be used to develop a customized object.) Due to assay requirements (e.g., 6-channel photometer), you must generate these compensation objects on the LightCycler 2.0 Instrument. The photometer in the LightCycler 2.0 Instrument can detect 6 different colors in distinct channels. The spectral distance of these channels is approx. 30 nm, much narrower than the nm distance in 3-channel LightCycler Instruments. Consequently crosstalk between channels is significantly higher; there may be signal bleed-over not only from a short wavelength into a longer wavelength, but also vice-versa (see Figure 3). Figure 3: Crosstalk between emission spectra on the 6-channel photometer 7

8 2.2 Color for Customized Applications continued Factors Influencing the Relative Crosstalk Values A multicolor experiment demands more stringent color compensation since it involves more colors and the distances between emission peaks of the fluorescent dyes are much shorter. In such specific applications crosstalk is also affected by the oligonucleotide sequences of the probes. However, as with dual color assays, the major factors influencing relative crosstalk values for multicolor experiments are: temperature properties of the optical system coupling chemistry of fluorescence dyes Applications Using Customized Dual Color Hybridization Probes Comments on Customized Dual Color HybProbe Assays Dual color hybridization probe assays usually use the combination of LC Red 640 and LC Red 705 fluorophores. For these, you can use a universal color compensation file or object created with the LightCycler Color Set. However, if you are using dual color assays with any other fluorophores (e.g., with LC Red 610 and LC Red 670), you must develop a color compensation object by the same procedure you would use for multicolor applications (as detailed below). Since this is a time-consuming process, we do not recommend customizing dual color hybridization probe assays. Instead, just use the LC Red 640/LC Red 705 dye combination Multicolor Hybridization Probe Applications Introduction Setting up a Multicolor Experiment If you use a multicolor application (e.g., multiplex PCR or multicolor detection), you must generate a parameter-specific color compensation object for each experiment, since the emission spectra are close to each other and can be influenced by each parameter. This is true for Roche Kits (which include reagents for color compensation) as well as for your own applications. Note: Color compensation is independent of final volume. If you need a color compensation object for an experiment with 100 µl samples, you can still use a 20 µl capillary to do the compensation experiment. To minimize the work involved, we recommend that you use the same probes in the color compensation experiment that you will use for detecting and analyzing the experimental samples. For customized multicolor compensation prepare capillaries containing: Blank For the blank, you can use water, but we highly recommend using a buffer (50 mm Tris, ph 8.3, with 0.25 mg/ml BSA). This buffer is also suitable for diluting and storing the primers and probes. Fluorescein-labeled probes as calibrator The final concentration of fluorescein-labeled probes in this capillary should be µm; use an equimolar mixture of all the different fluorescein-labeled probe sequences. 8

9 2.2.2 Multicolor Hybridization Probe Applications continued LC-Red-labeled probes as calibrators Each LC-Red-labeled probe should have its own capillary. (If you have more than one probe with the same LC-Red dye, use an equimolar mixture of all those probes in the same capillary.) Due to absence of a FRET partner, the signal intensity of the probe will be low. Therefore, use a final probe concentration of at least 1 µm. Note: You can include up to 6 capillaries (blank, Fluorescein, LC Red 610, LC Red 640, LC Red 670 and LC Red 705) in the run. Special Considerations Creating Color Objects Since the spectral distance between channels in the LightCycler 2.0 Instrument is short, channels might, in rare cases, pick up faint signals from neighboring channels, even after color compensation is activated. These signals are usually very small and are just slightly above background noise. To eliminate these artifacts, slowly reduce (by stepwise titration) the final concentration of Hybridization Probes in the color compensation run to the concentration used in the experimental assay (e.g., 0.2 µm each). Alternatively, use the non-automated analysis method (Fit Points) to manually raise the noise band. The multicolor compensation protocol is identical to that of the LightCycler Color Set. If you are an expert user, you may copy the macro from the Demo Macro folder (see 2.1.2), save it under a new name, and then change the settings in the Sample List (under Capillary View). Important: If a multicolor compensation object is to work properly, you must edit the analysisspecific sample list in the Color module. Most importantly, you must define the dominant channels (Figure 4). Figure 4: Using and editing a macro for multicolor color compensation Before starting the run, place the reagent capillaries into the carousel in the order listed above. Always place the capillary with buffer in the first position on the carousel. When the experiment is finished, select Color from the Analysis menu and then Save the CC Object (as in section 2.1.2). 9

10 2.2.2 Multicolor Hybridization Probe Applications continued Applying Color to a Multicolor Hybridization Probe Experiment You can now apply the parameter-specific color compensation object to the corresponding multicolor experiments. To activate the color compensation, click on Color in either the Run or the Analysis module, and choose the appropriate object under Select Color. Note: Select the appropriate channels for multicolor compensation, then click OK. Important: This color compensation will be valid for all experiments performed with the identical parameters. However, if you change the sequences of the hybridization probes, you will need to generate a new color compensation object. Even if you order a new batch of identical probes, it may be wise to re-run the color compensation experiment and compare the results obtained with the freshly generated ccc object to those obtained with the old ccc object. Automated Identification of Specific Color Objects by Using the "Color Comp ID Number The LightCycler SW version 4.0 and higher provides the option for an automated selection of a specific color compensation object. In the Capillary View under Samples there are several boxes offering room for additional run and/or sample information. The box for the color compensation identnumber (Color Comp ID) can be used to link an analysis file to the respective parameter-specific color compensation object. Especially when working with Macros, this ID number is highly convenient for the correct identification of color compensation objects. Example: Using the Demo Macro Color Set (Cat No ) for a dual color calibration run (LC Red 640 and 705), this Macro automatically labels the generated ccc.object with the Color Comp ID Any dual color run using this ccc.object can be automatically guided to this ccc.object by entering the identical number into the Color Comp ID box. The same function is available for customized applications. The ID box accepts any numbers, letters or letter-figure combinations. 10

11 3. Application Example Melting Curve Analysis without Color Figure 5: This example (from the LightCycler Multicolor Demo Set) shows a melting peak analysis in channel 670 of a target detected with the LightCycler Red 670 HybProbe set. Color was off, therefore no ccc object was applied to the results. Additional peaks seen in this channel arise from crosstalk generated by other probes that were labeled with LC Red 610, 640 and 705. These additional melting peaks prevent correct analysis of the data. Melting Curve Analysis with Color Figure 6: The identical data set from Figure 5 was analyzed in the Tm Calling module with Color on and the appropriate ccc object selected. The analysis of the target in channel 670 is now free of any interference from spectral overlap, and only the specific melting peak is visible. This data can now be accurately interpreted. Trademark: LightCycler is a trademark of a member of the Roche Group. The technology used for the LightCycler System is licensed from Idaho Technology Inc., Salt Lake City, UT, USA. SYBR is a registered trademark of Molecular Probes, Inc. 11

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