User manual

74 | Droplet Digital
™
PCR Applications Guide
Additional Applications
Demonstration of a milepost assay is presented in Figure 8.3, which shows the 2-D plots
of FAM amplitude and VIC amplitude for an RNaseP anchor assay (VIC), and progressively
farther assays on chromosome 10 for a human DNA sample. The upper left panel
demonstrates a control to account for the inherent probability of two copies completely
separated from each other (RNaseP, which is on chromosome 10, and an assay located
on chromosome 6) randomly co-localizing in the same droplet. The remaining panels
demonstrate that as the distance between the anchor and the milepost assay (on the same
chromosome as RNaseP) increases, the number of double-positive droplets decreases.
At a distance of 100 kb, the double-positive population is equal to the unlinked control.
Finer resolution could be done with more milepost assays to cover the range.
Fig. 8.3. Milepost assay results to determine the quality of DNA by a linkage study.
Channel 1 amplitudeChannel 1 amplitude
0 0–500 –1,000
0
0
0
0
500 1,000 1,0001,500 2,0002,000 2,500 3,0003,000 4,0003,500 5,000
12,000
8,000
12,000
8,000
10,000
7,000
10,000
7,000
8,000
8,000
6,000
6,000
5,000
6,000
6,000
4,000
5,000
4,000
4,000
4,000
3,000
2,000
1,000
2,000
3,000
2,000
2,000
1,000
Channel 2 amplitude Channel 2 amplitude
14,000
9,000
RNaseP/chromosome 6: FAM+VIC+ = 243
10 kb milepost: FAM+VIC+ = 793
1 kb milepost: FAM+VIC+ = 1,739
100 kb milepost: FAM+VIC+ = 230
microRNA Amplification by ddPCR
A method for absolute quantification of microRNA (miRNA) described here shows the
day-to-day reproducibility of a tenfold change in the starting quantity of synthetic template
mir-210. An example ddPCR amplification of an miRNA synthetic template (mir-210) was
done in a two-step reaction. The reverse transcription (RT) reaction was done in bulk
solution, and the cDNA was partitioned into droplets before PCR amplification.
miRNA synthetic template for mir-210 (RNase free, HPLC purified) was obtained from
Integrated DNA Technologies, Inc. The miRNA synthetic templates in TE buffer at a final
concentration of 1 µM were aliquoted into individual-use tubes and frozen at –80°C.
Each miRNA stock was loaded in RNase-free water on ice just before performing an RT
assay. Reverse transcription of the synthetic miRNA templates was done in bulk solution
using the TaqMan microRNA reverse transcription kit (Life Technologies Corporation),
and using the miRNA RT protocol with the specific RT primers for each miRNA.
0–1,000 1,000 2,000 3,000 4,000 5,000 0–1,000 1,000 2,000 3,000 4,000 5,000