User manual
Droplet Digital
™
PCR Applications Guide | 87
Additional Tips
No Concentration Calls on Some Wells
If a concentration estimate fails to appear in the concentration chart in QuantaSoft, this
indicates the software could not auto-analyze or assign droplets to positive or negative
populations using its auto-analysis algorithm, or the well had an unusually low droplet
count (<10,000). Low total droplet counts indicate a problem with the assembly of the
reaction mix, poor preparation of the sample, or poor handling. Manually set a threshold
and QuantaSoft software will calculate a concentration, which will appear in the
concentration chart.
Target Accessibility
Strong or excessive secondary structure can prevent a DNA target from being amplified.
Human gDNA and plasmid DNA can usually be restriction digested to remove inhibiting
secondary structure, thereby rescuing detection. RNA secondary structure is best
addressed by changing the location of the assay, if possible, or reverse transcribing the
assay at a warmer temperature.
Figure 9.8 is an example of poor target accessibility, manifested by the significant number
of mid-level amplitude droplets (that is, rain), which is resolved by performing a restriction
digestion on the DNA before ddPCR.
Fig. 9.8. Elimination of secondary structure allows efficient amplification and accurate quantification.
A, fluorescence amplitude plot showing four wells of undigested plasmid DNA (left) and four wells after restriction
enzyme digestion to linearize the plasmid (right); B, concentrations are corrected to the expected value after
restriction enzyme digestion (right) as compared to undigested samples (left).
Channel 1 amplitude
0
20,000
8,000
2,000
Event number
40,000 60,000 80,000
16,000
6,000
12,000
100,0000
A03 B03 C03 D03 E03
F03
G03 H03
4,000
10,000
14,000
A
Concentration, copies/µl
0
A03
3,000
Sample
7,000
2,000
5,000
1,000
4,000
6,000
B
1,490
5
B03 C03 D03 E03 F03 G03 H03
4
3
2
1
0
Copy number
1,600
1,610
1,520
6,170
5,940
6,170
5,620
Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting