Real-time PCR run times using GENAXXON MasterMixes are reduced by 30-50%, just by changing the settings of real-time PCR intsruments. A run time down to 41 minutes is obtained.
Fast real time PCR is widely requested by DNA laboratories in order to save time, to increase throughput and also to have increased instrument availability. Therefore, it is of interest to test the performance of GENAXXON MasterMixes using fast cycling conditions.
This study shows that GENAXXON ProbeMasterMix (2X) as well as GENAXXON GreenMasterMix (2X) used with fast cycling real- time 2-step protocols on standard real-time PCR cyclers can reduce runtime significantly.
Fast 2-step protocols were obtained by changing the settings on the three applied standard real-time PCR cyclers; LightCycler® 96, Mx3500P and StepOnePlus™. Each instrument exhibits different requirement for ROX reference dye levels.
Two different 2-step protocols were tested on each of the three real-time PCR instruments using two targets of different length; PAH12 (203 bp) and Pthr (75 bp). All six fast protocols tested show high precision and efficiencies. The obtained R2 and Cq values for the tested fast protocols are within the criteria normally set for the Genaxxon master mix standard protocol. All protocols and results are shown in table 1 – 3 and figure 1 – 6.
Conclusion and remarks:
Depending on the applied real-time PCR instrument and DNA target used, total run times of as fast as ~41 – 61 minutes were obtained. This is a significantly reduction compared to the standard protocol with a total run time of approximately 83 minutes.
The present study clearly demonstrates that it is possible to shorten real-time PCR run time of Genaxxon master mixes, severely, just by changing the standard real-time PCR protocols to 2-step protocols with short denaturation and elongation steps.
When performing fast real-time PCR, it is of high importance to consider primer design and target size. For efficient amplification during fast cycling conditions target size between 70 bp and 200 bp is recommended. The shorter the target length, the faster the total run time is. For other targets and other primer sets, the annealing step has to be determined experimentally.
Fast real-time PCR results using GENAXXON MasterMixes (without ROX) on LightCycler® 96
GreenMasterMix (2X) without ROX or ProbeMasterMix (2X) without ROX was tested on LighCycler96 (Roche) using fast cycling real-time 2-step protocols; Fast PAH12 Green LC and Fast Pthr Probe LC, respectively.
qPCR Setup: GreenMasterMix (2X) (without ROX) or ProbeMasterMix (2X) (without ROX), primers targeting either PAH12 (203 bp) or Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used. All DNA concentrations were tested in quadruple replicates. See fig. 1 and 2. The PCR reaction mix was run on LightCycler® 96 (No requirements for ROX). Instrument settings, run times and quality criteria for the applied fast protocols; Fast PAH12 Green LC and Fast Pthr Probe LC, were compared to the standard protocol; Standard Pthr LC. See table 1.
Results: The standard Fast Pthr Probe LC protocol has a run time of 80.8 minutes. Fast PAH12 Green LC and Fast Pthr Probe LC protocols, resulted in run times of 47.5 and 40.8 minutes, respectively. All protocol times are inclusive ramping time and a15 minutes hot start. The determined criteria, as shown in table 1, for Fast PAH12 Green LC and Fast Pthr Probe LC protocols were satisfying. Furthermore, the melt curve analysis using Green 2x Master Mix without ROX the Fast PAH12 Green LC protocol confirmed high specificity.
Fast real-time PCR results using GENAXXON MasterMixes (Low ROX) on Mx3005P
GreenMasterMix (2X), Low ROX or ProbeMasterMix (2X), Low ROX was tested on Mx3005P (Agilent Technologies) using fast cycling real-time 2-step protocols; Fast Pthr Green Mx and Fast Pthr Probe Mx, respectively.
qPCR Setup: GreenMasterMix (2X) (Low ROX) or ProbeMasterMix (2X) (Low ROX), primers targeting Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used. All DNA concentrations were tested in quadruple replicates. See fig. 3 and 4. The PCR reaction mix was run on MX3005P (Requiring Low ROX). Instrument settings, run times and quality criteria for the applied fast protocols; Fast Pthr Green Mx and Fast Pthr Probe Mx, were compared to the standard protocol; Standard Pthr Mx. See table 2.
Results: The standard Fast Pthr Probe Mx protocol has a run time of 83.4 minutes. Fast Pthr Green Mx and Fast Pthr Probe Mx protocols, resulted in run times of 50.1 and 46.8 minutes, respectively. All protocol times are inclusive ramping time and a15 minutes hot start. The determined criteria, as shown in table 2, for Fast Pthr Green Mx and Fast Pthr Probe Mx protocols were acceptable. Furthermore, the melt curve analysis using GreenMasterMix (2X), Low ROX Master Mix with Fast Pthr Green Mx protocol, confirmed high specificity.
Fast real-time PCR results using GENAXXON MasterMixes (High ROX) on StepOnePlus™
GreenMasterMix (2X) (High ROX) or ProbeMasterMix (2X) (High ROX) were tested on StepOnePlus™ (Life Technologies) using fast cycling real-time 2-step protocols; Fast PAH12 Green S1 and Fast Pthr Probe S1, respectively.
qPCR Setup: GreenMasterMix (2X) (High ROX) or ProbeMasterMix (2X) (High ROX), primers targeting either PAH12 (203 bp) or Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used. All DNA concentrations were tested in quadruple replicates. See fig. 5 and 6. The PCR reaction mix was run on StepOnePlus™ (Requiring High ROX). Instrument settings, run times and quality criteria for the applied fast protocols; Fast PAH12 Green S1 and Fast Pthr Probe S1, were compared to the standard protocol; Standard Pthr S1. See table 3.
Results: The standard Fast Pthr Probe S1 protocol has a run time of 85.9 minutes. Fast PAH12 Green S1 and Fast Pthr Probe S1 protocols, resulted in run times of 61.3 and 45.9 minutes, respectively. All protocol run times are inclusive ramping time and a 15 minutes hot start. The determined criteria, as shown in table 3, for Fast PAH12 Green S1 and Fast Pthr Probe S1 protocols were satisfying. Furthermore, the melt curve analysis, when using GreenMasterMix (2X) (High ROX) with the Fast PAH12 Green S1 protocol confirmed high specificity.
