User manual

Droplet Digital
™
PCR Applications Guide | 75
Additional Applications
Fig. 8.4. Day-to-day reproducibility of mir-210 miRNA assay: measured vs. theoretical.
Measured copies/µl
10.1
0.1
100 1,000 10,000
1,000
100
1
10
Estimated copies/µl
10,000
10
Day 1
Day 2
Multiplexing
Various schemes can be designed to add complexity to assays run in ddPCR. For instance,
you may want to add more than two assays to a reaction. The QX100
™
and QX200
™
Droplet
Digital PCR systems can detect fluorescence in two color channels (FAM and HEX).
However, there is an added dimension of fluorescence amplitude that can be used to
multiplex more than two assays at a time.
The basic concept for multiplexing is to make use of the differences in fluorescence
amplitude signal to change the spatial positioning of the droplet clusters in the 2-D data
plots drawn in QuantaSoft
™
software. By varying the concentration of the TaqMan assay,
the end-point fluorescence amplitude can be shifted in many assays. Due to this shift, you
can include two or more assays in the FAM and/or HEX channels and empirically determine
what concentrations will resolve the two assays in separate clusters.
ddPCR amplification of the cDNA generated in the RT reaction was done by adding 1.33 µl
of each cDNA to a 20 µl ddPCR reaction mixture containing 1 µl of 20x TaqMan miRNA
reagent (Life Technologies) specific to the miRNA of interest, 10 µl of Bio-Rad’s 2x ddPCR
supermix for probes, and 7.67 µl of molecular biology–grade water. The droplets were
generated, thermal cycled, and detected using standard procedures.
Day-to-Day Reproducibility Study: mir-210 miRNA
mir-210 synthetic templates at 1.6 nm originated from a single 1 µM stock. The 1.6 nM
stock was used to create nine concentrations (10 µl into 90 µl RNase-free water) on ice.
Concentrations 5–9 were used for the titration series, with an estimated concentration
range of 2,135–0.21 copies/µl of ddPCR. The data are plotted as individual well replicates
(duplicates) for each day with Poisson 95% confidence intervals in Figure 8.4.