Three different primer concentrations, 100 nmol/L, 200 nmol/L (the recommended starting concentration for UPL assays), and 300 nmol/L, and their combinations in a primer optimisation matrix were investigated, leaving all the other reaction conditions unchanged. A primer combination was considered to be optimal when the amplification resulted in an amplicon of the expected size where the following conditions were met; a low Cq value (the point where fluorescence intensity during amplification is significantly greater than background fluorescence), a low standard deviation between replicates, adequate signal to noise ratio in the sense that robust levels of fluorescence intensity were seen, and no (or very low levels of) primer dimers were present.
The RT-qPCR with the optimal primer concentration combination was further optimised with respect to the concentration of the probe. In preliminary experiments, a wider range of UPL probe concentrations were tested. Probe concentrations higher than 200 nmol/L did not significantly improve RT-qPCR assays. Furthermore, the cost of a probe-based RT-qPCR assay is strongly dependent on the cost of the probe. We therefore only used the concentrations 100 nmol/L and 200 nmol/L for further probe optimisation. The lower probe concentration was chosen except when the higher probe concentration gave a significantly lowered Cq value and/or resulted in a significant improvement of the signal intensity (signal to noise ratio).
We designed 63 RT-qPCR assays for a panel of DNA repair and reference genes using the web-based design service for UPL RT-qPCR assays as outlined in Material and Methods. An intron-spanning assay was not possible for eight (13%) of the genes. Two of these genes consisted of a single exon, whereas the software was only able to choose intra-exonic primers for the remaining six.
Sixty assays (95%) were successful after the use of the primer matrix to optimise the concentration of each primer. Three assays, did not meet the quality control criteria as they either gave multiple non-specific bands as seen on an agarose gel or gave poor amplification with all primer concentrations tested.
The Cq differences (primer combination showing the highest Cq value minus primer combination showing the lowest Cq value for a given RT-qPCR assay) observed for the 60 assays were in the range of 0.5 to 6.7. The observed Cq differences for a given RT-qPCR assay are due to the varying primer concentrations, as these were the only variable reaction parameters. The performance of the majority of the RT-qPCR assays were significantly dependent on primer concentration (Fig. 1A). Twenty seven assays (45%) had Cq value differences in the range of 1.1 to 2.0, and eight assays (13%) had a Cq value difference greater than 2.1. Twenty five assays (42%) that had Cq value differences in the range of 0.5 to 1.0 and were therefore less dependent on primer concentration.
The RT-qPCR products were also examined by gel electrophoresis before the optimal primer concentration combination was chosen to eliminate any conditions that gave unacceptable amounts of primer dimers.
The distribution of optimal primer concentrations is shown in Figure 1B. Thirty nine assays (65%) performed better with an asymmetric primer concentration combination, while symmetric primer concentrations performed better in 21 assays (35%).
As an example, the Cq values of the RT-qPCR assay for the NBS1 gene using different primer concentrations showed a difference of 5.5 (Fig. 2A). Each of the primer concentration combinations generated a specific amplicon as shown by gel electrophoresis (Fig. 2B) but 300 nmol/L of each primer performed best showing the lowest Cq value (Fig. 2A). In this case, the concentration of the forward primer had a greater contribution to higher RT-qPCR sensitivity than the concentration of the reverse primer (as seen in Fig. 2A).
After the optimal primer combination was chosen, we optimised the concentration of the probe for that particular primer combination. Forty seven of 60 assays (78%) were found to be optimal with 100 nmol/L, whereas 13 assays (22%) performed better with a probe concentration of 200 nmol/L.
When a default concentration of 200 nmol/L for each primer was used, a satisfactory result was observed in 54 out of 63 assays (86%). Nevertheless, only seven assays out of 60 (12%) performed best using a primer combination of 200 nmol/L for each primer (Fig. 1B). This makes it clear that optimising a new RT-qPCR assay is essential to guarantee its efficiency as well as its specificity.
The range of concentrations to test in the primer optimisation matrix as well as the concentrations of the probe to use are likely to be dependent on the amplification monitoring system used and needs to be determined for each system separately.