Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays

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.

[1]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[2]  Philip Lijnzaad,et al.  The Ensembl genome database project , 2002, Nucleic Acids Res..

[3]  Carl T. Wittwer,et al.  Rapid Cycle Real-Time PCR — Methods and Applications , 2002, Springer Berlin Heidelberg.

[4]  Tania Nolan,et al.  Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. , 2004, Journal of biomolecular techniques : JBT.

[5]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[6]  Wolfgang Vahrson,et al.  Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability , 2005, Nucleic acids research.

[7]  R E Rhoads,et al.  Optimization of the annealing temperature for DNA amplification in vitro. , 1990, Nucleic acids research.

[8]  D. Ginzinger.  Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. , 2002, Experimental hematology.

[9]  Alfred Pingoud,et al.  Real‐Time Polymerase Chain Reaction , 2003, Chembiochem : a European journal of chemical biology.

[10]  Niels Tolstrup,et al.  The ProbeLibrary™ - Expression profiling 99% of all human genes using only 90 dual-labeled real-time PCR Probes , 2004 .

[11]  E Schütz,et al.  Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. , 2001, Clinical chemistry.

[12]  Lyndon Gommersall,et al.  Basic principles of real-time quantitative PCR , 2005, Expert review of molecular diagnostics.

[13]  Tania Nolan,et al.  Quantification of mRNA using real-time RT-PCR , 2006, Nature Protocols.

[14]  R. Rasmussen.  Quantification on the LightCycler , 2001 .

[15]  J. Robertson,et al.  An introduction to PCR primer design and optimization of amplification reactions. , 1998, Methods in molecular biology.

[16]  Carl T. Wittwer,et al.  Rapid Cycle Real-Time PCR — Methods and Applications , 2002, Springer Berlin Heidelberg.