The MyGo Pro instrument acquires data through 121 channels which allows the software to display spectral data graphically. This data can be useful when trying to differentiate between differently labelled dyes that emit fluorescence at different wavelengths. In more challenging applications like multiplexing, this can be very useful when balancing oligo concentrations in reactions involving multiple different probes labelled with different dyes.
Performing Spectral View
Spectral View can be performed on any experiment that has acquired fluorescent data, either during cycling or melting. However, Targets do not have to be assigned to samples in order to detect spectral data. Open up the “MyGo Pro Spectral View.ppf” experiment file from the MyGo Pro walkthrough folder.
Select Samples. Now select Samples as Plate if not already selected.
In this experiment there are four samples. The red sample only contains a FAM reaction, the green sample only contains a CAL 540 reaction and the purple sample only contains a CAL 560 reaction. The blue sample contains FAM, CAL 540 and CAL 560 in one reaction. In the Targets pane we can see that there are three differently labelled targets that have been assigned to all wells.
Select Analysis Type
Click the Analysis tab, Manual and add a Spectral View analysis from the Select Analysis Type window. If not already present learn how to do this in the Select Analyses Type section.
Wells as Table
Click the Wells as Table tab, if it is not already selected. By default, A1 is the well that is selected when opening up this experiment.
It is possible to view spectral data that is acquired during different programs throughout the experiment. By default, all programs are selected. In this example select the 2-Step Amplification program as shown below:
Samples as Plate
The Samples as Plate view shows the features described in Working with Plate Displays.
The Spectral Graph occupies the top half of the Spectral View analysis pane as shown in the example above. It is the graphical representation of spectral data that is acquired during a specific program, chosen in the Programs tab.
During a PCR experiment each cycle acquires data during an acquisition. In this example 45 cycles were performed, therefore 45 acquisitions were taken. Each line on the spectral graph will represent every one of these acquisitions. During early PCR cycles the fluorescence intensity will be relatively low and constant. However during amplification, fluorescence will increase which will produce a spectral line that has a larger intensity at the wavelength the fluorescence is emitting. Every subsequent cycle, assuming amplification continues, will produce an additional line, higher than the previous. Subsequent line intensities on the spectral graph represent increases in fluorescence acquired during subsequent PCR cycles.
Spectral Graph Controls
There are a number of controls which manipulate the way you can see the data presented in the Spectral Graph. These are highlighted below:
Display All Acquisitions Enabled
This option allows the user to Display all acquisitions. In the example above this has been turned on and therefore all 45 acquisitions are present on the graph.
Display All Acquisitions Disabled
When this has been disabled the acquisition displayed on the Spectral Graph will correspond to the acquisition programmed into the Display Acquisition Index box.
Display Acquisitions Index
An acquisition index corresponds to the cycle that a specific acquisition was taken. In the example below Display Acquisition Index 45 has been entered. However, to display this acquisition on its own Display all acquisitions must be disabled, as shown in the example below:
For the purpose of this section enable Display all acquisitions. Inevitably, everything emits fluorescence somewhere in the visible spectrum, even the optics unit used to read fluorescent data from a reaction. The Subtract background control removes any background fluorescence, determined within the first few cycles, from the fluorescent data acquired after these cycles. When this control is disabled, early acquisitions no longer start from zero as shown below.
Individual Spectral Curves
In Samples as Plate select wells A1, B1 and C1, making sure Subtracted Background has been enabled as shown below.
As you can see, three differently colored spectral curve sets are displayed on the Spectral Graph. These probes are labelled with dyes which have different fluorescence emission spectra which helps deconvoluting the dyes from one another.
Combined Spectral Curve
In Samples as Plate select D1 to view a reaction that contains a combination of FAM, CAL 540 and CAL 560, as shown below.
To compare these against the individual spectral curves select all wells in column 1 as shown below. Notice that combined spectral data is larger than individual spectral curves. This is useful when needing to balance probe concentrations between differently labelled targets.
This concludes the section on Spectral View.