Quantitation methods
Before performing digital PCR experiments, consider quantifying the amount of
gDNA or cDNA in each sample.
We recommend the following methods of quantitation:
• Quant-iT
™
assay nucleic acid quantitation using the Qubit
™
Quantitation Platform
or
• Use spectrophotometer to determine nucleic acid concentration
Should a target be present at a sufficiently high concentration in the sample of
interest, it is possible that all reaction replicates will be positive, thus preventing the
determination of the target concentration. In this case, the sample must first be
diluted prior to running the digital PCR experiment.
In a dPCR experiment, gDNA samples are diluted down to a limiting quantity, such
that most individual PCR reactions contain either zero or one target molecule. The
procedure for determining the optimal dilution for a sample differs depending on
whether or not the target copy number per genome is known.
If the target copy number per genome of your samples is known, dilute the samples
so that each reaction well on a chip will contain approximately 0.6 to 1.6 copies of
the target sequence. For example:
1. Assuming 3.3 pg/copy of a given gene in the human genome, and a reaction-
well volume of 755 pL
2. Dilute the stock gDNA in a given sample to ~1,045 copies/μL or 3.45 ng/μL in
the final reaction, resulting in ~0.73 copies per reaction well.
Determine the target copy number per genome
To determine the copy number per genome:
1.
If the source or species of the gDNA is known , refer to
to determine the size of the genome.
2.
Once the size of the genome is known, calculate the mass using the following
formula:
m
= (
n
) ( 1.096 × 10
-21
g/bp )
where
m
is the genome mass in grams, and
n
is the genome size in base pairs.
The following example calculates the mass of the human genome using the Celera
Genomics estimate of 3.0 × 10
9
bp (haploid):
m
= (3.0 × 10
9
bp) (1.096 × 10
-21
g/bp)
m
= 3.3 × 10
-12
g or 3.3 pg
The example is relevant to any gene that is present at the “normal” rate of two copies
per diploid genome, such as RNase P, and provides a basis to perform a digital
screening experiment to determine the optimal digital range.
Sample dilution
Determine the
optimal dilution
when the
target is known
Chapter 3
Prepare the digital PCR reaction and load the chips
Prepare the DNA samples
3
QuantStudio
™
3D Digital PCR System User Guide
21
