iScript™ IV SYBR Green One-Step RT-qPCR Kit

iScript™ IV SYBR Green One-Step RT-qPCR Kit is a complete system containing all necessary reagents for both reverse transcription and PCR amplification to occur in a single reaction tube using gene-specific primers. RT-qPCR Enzyme Mix contains iScript™ IV Reverse Transcriptase (RT), Hot-Start Taq DNA Polymerase and RNase Inhibitor for highly sensitive and specific RT-PCR using any RNA template. Our proprietary RT-qPCR Master Mix contains stabilizers and enhancers that optimize the activities of both reverse transcriptase and Taq DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR artifacts.

The system enables highly sensitive detection from as few as 10 copies of a target gene, with a broad dynamic range that supports accurate quantification of high-copy mRNA from up to 1 μg of total RNA.

• iScript IV Reverse Transcriptase is a new version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can synthesize cDNA at a temperature range of 42–60°C.
• AccuStart Taq DNA polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks polymerase activity at ambient temperatures. Activity is restored after the denaturation step in PCR cycling, providing an automatic “hot start” in PCR for increased sensitivity, specificity, and yield.
• RT-qPCR Master Mix consists of a proprietary buffer system, SYBR Green I, MgSO₄, dNTPs, and stabilizers. SYBR Green I is a fluorescent dye that binds directly to double-stranded DNA. In qPCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and that can be detected using real-time qPCR instruments. SYBR Green I in this formulation can quantify as few as 10 copies of a target gene in as little as 0.1 pg of total RNA, has a broad dynamic range of six orders of magnitude, and is compatible with melting curve analysis.

RNase Inhibitor is included in the enzyme mix to safeguard against degradation of target RNA due to ribonuclease contamination. ROX Reference Dye is included as a separate component to normalize the fluorescent signal between reactions.

Applications

• Gene-expression analysis.
• Transcription analysis.
• Gene cloning.
• Virus detection and quantification.

General Protocol

RT-PCR reactions should be assembled in a nuclease-free environment. The use of clean pipettes designated for PCR and aerosol resistant barrier tips are recommended.

1. Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes.

2. Prepare the following reaction mixture in a qPCR tube on ice:
     

3. Mix carefully by vortexing for 3-5 seconds. Centrifuge briefly to collect the contents of the tube.

4. Program the thermal cycler so that cDNA synthesis is followed immediately by qPCR amplification.
     

Results: