TB Green Premix Ex Taq (Tli RNase H Plus) master mix is optimized for efficiency and versatility, providing superior results when low yields or no amplification is obtained with other enzymes. In this experiment, Nox4 (NADPH Oxidase 4) mRNA expression was analyzed by qPCR using TB Green Premix Ex Taq master mix. Previous attempts to analyze this target using a SYBR green master mix from another supplier were unsuccessful. However, amplification with TB Green Premix Ex Taq master mix provided reliable results without any reaction optimization.
Tech Note
qPCR without optimization using TB Green Premix Ex Taq
Data kindly provided by: Hong Seok Kim, Postdoctoral researcher, UT Health Science Center at San Antonio
Introduction
Results
Nox4 expression in THP-1 monocytes was analyzed by RT-qPCR. TB Green Premix Ex Taq master mix was used to amplify a portion of Nox4 from cDNA. Amplification curves were obtained using template cDNA equivalent to 10 and 100 ng of RNA (Figure 1), confirming successful amplification of the Nox4 target.
Figure 1. qPCR amplification curves for Nox4. Amplification was performed with 10 and 100 ng of cDNA template (RNA equivalent). The no-template control was not amplified. The resulting Ct< values are shown in the orange box.
Conclusions
The difficult-to-analyze Nox4 mRNA could be measured by qPCR with TB Green Premix Ex Taq master mix. Previous attempts to analyze Nox4 using a SYBR green master mix were unsuccessful, but amplification using TB Green Premix Ex Taq master mix provided reliable results the first time.
Methods
Total RNA was prepared from THP-1 monocytes using a commercially available RNA extraction kit (Ambion). cDNA was synthesized by reverse transcription with the QuantiTec Reverse Transcription Kit (Qiagen) following the manufacturer's recommended protocol. The cDNA was used for intercalating green dye-based qPCR analysis of a 281-bp portion of Nox4 using TB Green Premix Ex Taq (Tli RNase H Plus) master mix on an ABI 7900HT system (Thermo Fisher Scientific). Reactions were assembled according to the recommended protocol (Table I). Reactions were performed in duplicate. The PCR amplification conditions were 95°C for 30 sec (initial denaturation), then 40 cycles of 95°C for 5 sec, and 60°C for 34 sec.
| Component | Amount |
|---|---|
| TB Green Premix Ex Taq (Tli RNaseH Plus) (2X) | 10 µl |
| Forward Primer (10 µM) | 0.4 µl |
| Reverse Primer (10 µM) | 0.4 µl |
| ROX Reference Dye (50X) | 0.4 µl |
| Template | 2.0 µl |
| Sterile dH2O | 6.8 µl |
| Total | 20 µl |
Table I. qPCR reaction composition.
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