Techne PrimeQ - Manual (Page 162)

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3.19 Multiplex assays
Multiplex assays (or multi-colour detection) combine the use of two or more different reporters in
the same well. PrimeQ’s cartridge carousel can house up to four pairs of excitation and emission
filters, and as such, is capable of detecting up to four different reporters.
Multiplexing is suitable for all analysis methods as long as different reporters are used with
different emission spectra and that if more than one PCR is occurring in the same well, the
reactions do not compete. Furthermore, if the results are to be compared, the reactions should
also have similar efficiencies. These are recommended to be in the range of 1.95 to 2.05. Multiplex
assays are useful when analysing against internal controls, interpreting duplex PCR runs or in
mutation analysis as detailed in Allelic discrimination (section 4.9).
Multiplexing is a cost-effective approach, bringing savings in terms of reagents, sample and block
space. The method also helps when comparing results, as reactions are subjected to the same
PCR conditions. An associated disadvantage however is that optimizing a multiplex assay is
frequently a time-consuming process.
Example applications include:
Relative quantification
This analysis method compares the concentration of two reporters in a single well. The experiment
is designed in the same way as the basic quantification assay but allows the comparison of two
different reporters in the same well. This cuts down on reagents and allows more reliable
comparison between the two targets as reaction conditions are guaranteed to be the same and the
amount of starting material is the same. This approach requires the use of standards.
Allelic discrimination
Capable of detecting single nucleotide differences, this analysis method can only be used if there
are two reporters. Probes specific for allele 1 and allele 2 are labelled with different reporter dyes
and as mismatches between a probe and target will reduce the efficiency of the hybridization, this
will be reflected in the relative fluorescent signal post-PCR. Comparing the relative fluorescence of
each reporter will indicate whether the DNA sample is homozygote or heterozygote for allele 1 and
2 (see section 4.9.3.1).
Internal controls
A second set of primers and reporter labelled probe are used to amplify a second DNA template
which is spiked into each PCR reaction. The control serves as a performance indicator for the
PCR and is particularly useful in plus-minus scoring to guard against negative calls when a PCR
has simply failed, or there is an inhibitor in the sample.
3.19.1 Multiplex setup
To perform a multiplex assay, two or more reporters must be defined in the Program.
• Define the thermal cycling parameters as detailed in section 3.4.3.
• Add a dye read to the required step as detailed in section 3.4.3.5.
• Add further reads to the step by clicking on the Add Read button. The reads will appear as
tabs in the step settings box.
Refer to the relevant sections in Chapter 4 for detailed information on the analysis setup for
individual methods.
Having assigned multiple reporters in the program setup, these will now be available when setting
up the analysis method.
Note that the setup and analysis parameters will be consistent between dyes; if the user wishes to
analyse dyes differently, then the analysis settings must be changed post-run from the Result
Editor.