User manual

Droplet Digital
™
PCR Applications Guide | 31
Copies per Microliter
QuantaSoft software provides concentration results in copies of target per microliter
(copies/µl). The dynamic range of ddPCR extends from fewer than 0.25 copies/µl to more
than 5,000 copies/µl. In many cases, the fundamental quantity of interest to a user is the
number of copies of target in the starting sample.
The following example shows how copies/µl is converted to copies in the starting material.
Mix 10 µl of sample with 12.5 µl of ddPCR supermix for probes and 2.5 µl of assay
(primer and probe mix), for a total volume of 25 µl. Load 20 µl of this mix into a DG8
™
droplet
generator cartridge and run ddPCR. The software reports that the concentration is 8 copies/µl.
Two equivalent methods illustrate how many total copies and how many copies/µl of the
target DNA were present in the original 10 µl sample.
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Method #1: The ratio of sample to total volume is 10/25 = 2/5. Since there were
8 copies/µl in the final PCR mix, there were 8 x (5/2) = 20 copies/µl in the original sample.
In the full 10 µl of the original sample, there were 10 x 20 = 200 copies of the target DNA
■
Method #2: Since there were 8 copies/µl in the PCR mix and a total of 25 µl of the PCR
mix was made, there were 8 x 25 = 200 copies of the target DNA in the PCR mix. This
mix contained 10 µl of the original sample, so there were 200 copies of target DNA in the
full 10 µl of starting sample, and 200/10 = 20 copies/µl of target in the starting sample
Copies per Droplet
A useful unit for discussing the calculations underlying ddPCR is copies of target per droplet
(CPD). For illustration purposes in this chapter we will use 1 nl volume.
CPD represents a number per unit volume, and not a mass per unit volume. CPD is the
average number of target copies/droplet. For a CPD of 2, for example, some droplets will
have 0 copies, some will have 1, 2, 3, and so on.
There are multiple ways to calculate CPD:
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Example 1:
CPD = total number of molecules/total number of droplets
If you have 100,000 molecules total and 20,000 droplets, then
CPD = 100,000 molecules/20,000 droplets
CPD = 5 molecules/droplet
■
Example 2:
CPD = (molecules/µl) x droplet volume (µl)
If you have 20 molecules in a 20 µl sample with 1 nl droplet volume, then
20 molecules/20 µl = 1.0 molecule/µl
CPD = (1.0 molecule/µl) (0.001 µl/droplet)
CPD = 0.001
Absolute Quantification and the Statistics of Droplet Digital
™
PCR