To generate standard curves for the selected primers and the reference primers a log10 dilution series of genomic DNA was prepared at concentrations ranging from 102 nM to 10-2 nM. Each dilution was tested in triplicate. When analyzed by qPCR, the dilution series produced a set of standard curves, which were used to calculate the slope value with the aid of the SDS software version 2.1 Applied Biosystems (values are shown in Table 1). See Additional File 3, Figure 2 e. and f. for an example of SDS output report of standard and amplification plots.
Each qPCR experiment contained triplicates of the no-template-controls and patient samples for all of the primers tested. On the same reaction plate all DNA samples were tested with the test and reference primers. When any particular sample was being tested, the qPCR using reference primers and that sample were always included on the reaction plate. Each experiment was performed in triplicate, with replicates being performed on different days. Quantification was based on the increased fluorescence, which was measured and recorded using the ABI Prism 7900 sequence detection system and associated SDS software version 2.1 (Applied Biosystems). Results were expressed in terms of the threshold cycle value (Ct; the cycle at which the change in fluorescence for the SYBR dye passes a significance threshold). The threshold values are shown in Table 1. The output of the results was exported in tab-delimited text file format. Further calculations were performed using Microsoft Excel. PCR products were resolved by agarose gel electrophoresis to confirm the presence of a single band of the expected size (See Additional File 3, Figure 2 a. showing SDS output of amplification plot for 14 DNA samples for the PRODH and G6PDH primer sets along with images of corresponding gel bands c. and d.).
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