Abstract
BackgroundThe development of reverse transcription – quantitative real-time PCR (RT-qPCR) platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Therefore, we investigated the impact of varying the primer concentration, leaving all the other reaction conditions unchanged, on a large number of RT-qPCR assays which in this case were designed to be monitored using hydrolysis probes from the Universal Probe Library (UPL) library.FindingsOptimal RT-qPCR conditions were determined for 60 newly designed assays. The calculated Cq (Quantification Cycle) difference, non-specific amplification, and primer dimer formation for a given assay was often dependent on primer concentration. The chosen conditions were further optimised by testing two different probe concentrations. Varying the primer concentrations had a greater effect on the performance of a RT-qPCR assay than varying the probe concentrations.ConclusionPrimer optimisation is important for improving the performance of RT-qPCR assays monitored by UPL probes. This approach would also be beneficial to the performance of other RT-qPCR assays such as those using other types of probes or fluorescent intercalating dyes.
Highlights
The development of reverse transcription – quantitative real-time PCR (RT-qPCR) platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions
In order to set up multiple RT-qPCR assays for gene expression profiling it is necessary to run them at common thermal cycling parameters, which precludes assay optimisation by varying the annealing temperature
Whereas the principles of good primer design have been stressed for those performing RT-qPCR assays using an intercalating fluorescent dye, the use of probe-based assays has often led to the belief that the specificity of the probe means that less care is necessary with primer design and assay optimisation [10]
Summary
The development of reverse transcription – quantitative real-time PCR (RT-qPCR) platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Reverse transcription – quantitative real-time PCR (RTqPCR) is the preferred method to quantify RNA when a wide dynamic range and high signal to noise ratios are desired. A robust, reproducible, and optimised RT-qPCR assay is one of the key requirements for reliable gene expression data. RT-qPCR optimisation has become disregarded by many research groups in the era of high throughput analysis and rapid data reporting [2]
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