qPCR Results without Optimization with SYBR Premix Ex Taq
Data kindly provided by: Hong Seok Kim, Postdoctoral researcher (UT Health Science Center at San Antonio)
SYBR Premix Ex Taq 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 SYBR Premix Ex Taq. Previous attempts to analyze this target using a SYBR green master mix from another supplier were unsuccessful. However, amplification with SYBR Premix Ex Taq provided reliable results without any reaction optimization.
Nox4 expression in THP-1 monocytes was analyzed by RT-qPCR. SYBR Premix Ex Taq 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 from 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.
The difficult-to-analyze Nox4 mRNA could be measured by qPCR with SYBR Premix Ex Taq. Previous attempts to analyze Nox4 using another SYBR green master mix were unsuccessful, but amplification using SYBR Premix Ex Taq provided reliable results the first time.
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 SYBR green-based qPCR analysis of a 281 bp portion of Nox4 using SYBR Premix Ex Taq on an ABI 7900HT system (Life Technologies). 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.
Table I. qPCR reaction composition.