TB Green Premix Ex Taq II (Tli RNase H Plus) is ideal for real-time PCR experiments where specificity is critical. A high level of precision is achieved and maintained across samples through effective suppression of amplification of primer dimers and off-target products. This reagent is recommended for situations where primer design has been optimized, but increased qPCR specificity is still needed.
In this study, the mRNA levels of transcription factor E2F1 were analyzed in HEK293 cells transfected with either an experimental or control plasmid. The protocol was carried out with TB Green Premix Ex Taq II (Cat. # RR820Q) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific), using a 30-second extension step.
Results
Melt curve analyses indicated a high specificity for both E2F1 and the GAPDH control (Figure 1, bottom panel). A relative quantification of the raw data was performed using the delta delta Ct method. This analysis (Figure 2) showed no significant difference in E2F1 mRNA levels between samples, indicating the capacity of the TB Green master mix to provide reliable results with low sample-to-sample variation.
Figure 1. Amplification curve (top) and melt curve (bottom) for E2F1 and GAPDH (endogenous control).
Figure 2. Relative quantification of E2F1 mRNA levels of HEK293 cells transfected with the control plasmid and the focused gene A plasmid.
Conclusions
The use of TB Green Premix Ex Taq II resulted in high specificity and low sample-to-sample variation in real-time PCR results. This premix provided consistent results under the conditions tested.
Methods
HEK293 cells were transfected with a control vector or a focused gene A plasmid, and RNA was isolated using Trizol Reagent (Thermo Fisher Scientific). First-strand cDNA (localizing to the cDNA of E2F1) was generated using the RT2 First Strand Kit (SABiosciences), according to the manufacturer's instructions.
The first-strand cDNA was used as template for amplification of a 150-bp fragment (corresponding to the E2F1 cDNA) via real-time PCR with TB Green Premix Ex Taq II. GAPDH was used as an endogenous control. Reaction mixtures were prepared as described in the user manual.
After reaction assembly, a StepOnePlus Real-Time PCR System was used for real-time PCR using the recommended cycling conditions in the TB Green Premix Ex Taq II user manual.
