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DTI FabGreen HiFid Taq HS Premix minimizes PCR inhibition from dsRNA/cDNA hybrids, a common issue with low or RNase H minus RTs, GC-rich templates, or low-expression genes. It prevents poor qPCR amplification or reaction failure without extra cost or the need for a separate RNase H digestion step.

Specifications

Documents

Applications

Brand	Catalogue Number	Pack Size
DTI Green HiFid Taq HS Premix	DT0602.100	100 rxn (20 μl volume)
DTI Green HiFid Taq HS Premix	DT0602.500	500 rxn (20 μl volume)

• Lower Cq values as a result of fast extension rates

• Use with broad range of amplicon sizes, even over 500 bp

• Convenient 2X master mix is excellent for high-speed qPCR

• High sensitivity: detects fewer than 100 copies

• Hot-start PCR enzyme enables high efficiency and sensitivity in real-time PCR (qPCR)

• Compatible with all commonly used real-time PCR (qPCR) instruments

• Minimizes qPCR inhibition due to residual mRNA in input cDNA

• Includes DTI Green for intercalator-based qPCR

DTI Green HiFid Taq HS Premix - Protocol

DTI Green HiFid Taq HS Premix - Certificate of Analysis

DTI Green HiFid Taq HS Premix - Promotional Document

Results

qPCR without optimization using DTI Green HiFid Taq HS Premix

Nox4 expression in THP-1 monocytes was analyzed by RT-qPCR. DTI Green HiFid Taq HS Premix 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 DTI Green HiFid Taq HS Premix master mix. Previous attempts to analyze Nox4 using a SYBR green master mix were unsuccessful, but amplification using DTI Green HiFid Taq HS Premix 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 DTI Green HiFid Taq HS Premix (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

DTI Green HiFid Taq HS Premix (Tli RNaseH Plus) (2X)

Forward Primer (10 µM)

Reverse Primer (10 µM)

ROX Reference Dye (50X)

Template

Sterile dH2O

Total

Amount

10 µl

0.4 µl

2.0 µl

6.8 µl

20 µl

Table I. qPCR reaction composition.

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