For a target FFPE DNA sample, 5 ng of template (nominally determined by NanoDrop) was input into QFI-PCR, and the Cq output was converted to a 'functional' copy number.
A Danish template was used and inputs were received from Germany, Belgium, United Kingdom, Sweden, France, Luxembourg, Italy, Estonia, Czech Republic, Slovakia, Portugal and Cyprus.
By these means two independent proof-of-principle studies [ 11, 33] indicated that digital PCR could be used for reliable discrimination between aneuploid and euploid cases on pure fetal genetic material, and that this may be possible when only 10% of the input template was of aneuploid origin, provided that 4,000 individual events were monitored [ 11, 32, 33].
The same primers as those described for Conv-RT-PCR were used for the RT-qPCR analysis where the input RNA template was not subjected to DNase I treatment.
Although the input of template was standardised at 10 ng of DNA, the Ct values for the housekeeping genes differed slightly from patient to patient and from group to group (Table 3), thus demonstrating the need for correction of the raw data prior to further analysis.
Once the optimal anchored target-template alignments were computed, these were input to the automated homology modeling script of Discovery Studio from Accelrys, Inc. (San Diego, CA) that uses standard force fields to determine the energy minimized 3-D structural coordinates for the test sequences, including those from recent CASP experiments (as illustrated by examples in Figure S7).
After hybridization, the probes specific to human genomic DNA and the input template are ligated at the same time by a thermally stable ligase enzyme.
Unlike real-time PCR, where an 'analogue' signal of the entire PCR reaction containing the entire input template is obtained, in 'digital PCR' the PCR reaction is split into thousands of minute individual reactions, with each individual reaction containing at most a single template copy.
In our experience, even a minute amount of leftover BC primers can risk dimer formation with non-BC primers, as well as causing a "barcode resampling" problem, i.e. the same DNA input template being associated with multiple molecular barcodes, which defeats the benefits of molecular barcoding.
The input buffer template is filled from the moment the metronome - indicating the predefined beat-pattern and tempo - is activated.
