One-Step RT-PCR System

The innovative technology used in the SuperScript IV UniPrime One-Step RT-PCR System does not require T_m optimization for each primer pair. A universal annealing temperature of 60°C is suitable for most primer pairs, helping minimize optimization steps, save time, and avoid mistakes in reaction setup (Figure 1).

Figure 1. One-step RT-PCR cycling under two annealing conditions. Nine targets were amplified from 10 ng Universal Human Reference RNA (UHRR); the first batch was amplified using a universal annealing temperature of 60°C (left), and the second batch was amplified using annealing temperatures calculated with the T_m calculator for Platinum SuperFi DNA Polymerase (right). The molecular weight marker is Thermo Scientific GeneRuler 1 kb Plus DNA Ladder, ready-to-use.

The SuperScript IV UniPrime One-Step RT-PCR System is highly sensitive and detects low-abundance targets (Figure 2); this allows for one-step RT-PCR experiments with limited RNA input.

Figure 2. Target detection from low amounts of input RNA. Amplification of 0.43 kb fragment from serial dilution from 1 μg to 0.01 pg of UHRR with SuperScript IV UniPrime One-step RT-PCR System. The molecular weight marker is Thermo Scientific GeneRuler 100 bp Plus DNA Ladder, ready-to-use.

The SuperScript IV UniPrime One-Step RT-PCR System withstands the effect of common RT and PCR inhibitors such as copurified compounds from biological samples or reagents used in RNA purification. This exceptional robustness makes the system less dependent on RNA sample purity to help achieve reliable results. The enzymes in other one-step RT-PCR kits were inhibited by the presence of inhibitors (Figure 3).

Figure 3. Resistance to inhibitors. Detection of a 1 kb RNA target from 100 ng UHRR using the SuperScript IV One-step RT-PCR System or other one-step RT-PCR kits in reaction mixtures containing: (1) no inhibitor, (2) xylan (2.5 μg/μL), (3) humic acid (15 ng/μL), (4) SDS (0.013%), (5) Trozol (1.2%), or (6) LiCl (2.5 μg/μL).

With the innovative two-phase hot-start activation mechanism, the SuperScript IV UniPrime One-Step RT-PCR System allows for room temperature reaction setup and stability of preassembled reactions for an extended time. Highly efficient and specific amplification is observed even after prolonged storage at room temperature.

• For targets up to 3 kb, successful amplification is observed after reactions are left for 24 h at room temperature (Figure 4A)
• For longer targets, reactions can be stored for up to 4 hours (Figure 4B)

Combination of highly processive enzymes in the SuperScript IV UniPrime One-Step RT-PCR System allows for the amplification of a broad range of target lengths (Figure 5). Full length cDNA is synthesized in as few as 10 min, and the PCR step requires an extension time of only 30 sec/kb, resulting in one of the shortest protocols among one-step RT-PCR kits.

Figure 5. Versatility across a broad range of target lengths. Detection of RNA fragments ranging from 0.2 to 9.4 kb with the SuperScript IV UniPrime One-Step RT-PCR System and other one-step RT-PCR kits.

In colored format, the SuperScript IV RT Mix contains red tracking dye and the UniPrime RT-PCR Master mix contains blue tracking dye. When the two solutions are mixed during reaction setup, the final reaction mix turns purple, allowing for visual tracking of the reaction setup to prevent pipetting errors (Figure 7).

Yes. SuperScript IV UniPrime One-Step RT-PCR System works at both universal 60°C annealing temperature and at annealing temperature calculated with a T_m calculator.

Good laboratory practices are important for long fragment one-step RT-PCR. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Optimization steps to consider include longer extension times as recommended in the protocols and increasing template amounts. Learn more about RT-PCR reaction optimization and setup by visiting our reverse transcription educational resources.

With the SuperScript IV UniPrime One-Step RT-PCR system, cDNA synthesis can be performed at higher temperatures than with many other one-step RT-PCR products. For GC-rich or structurally complex RNA templates, it is recommended to increase the cDNA synthesis incubation temperatures up to 55–65°C.

The two-phase hot-start mechanism, utilized in SuperScript IV One-Step RT-PCR System and SuperScript IV UniPrime One-Step RT-PCR System, ensures sequential activation of RT and PCR enzymes in the one-step RT-PCR workflow. At ambient temperature, SuperScript IV RT remains inactive with a heat-sensitive RT-blocker. During the first hot-start activation phase at approximately 45°C, the RT-blocker is released, and the first-strand cDNA synthesis is initiated. During the second activation phase, the reaction is heated to 98°C to activate DNA Polymerase and simultaneously inactivate SuperScript IV RT. This mechanism separates the RT and PCR enzymes’ activities, delivering the highest RT-PCR specificity and yield.

The SuperScript IV UniPrime One-Step RT-PCR System produces blunt-end PCR products that can be cloned directly into blunt-end cloning vectors. TA cloning is also possible if 3′ dA-overhangs are added after PCR. Learn more about the use of different PCR enzymes for cloning application by visiting PCR educational resources.

Red and blue tracking dyes included in the colored format of SuperScript IV UniPrime One-Step RT-PCR System do not interfere with electrophoresis in E-Gel agarose gels. For optimal separation using E-Gel precast agarose gels, dilute the sample 2- to 30-fold, following the recommendations for specific E-gel.

SuperScript IV UniPrime One-Step RT-PCR System includes 2X UniPrime RT-PCR Master Mix containing modified Invitrogen Platinum SuperFi II DNA Polymerase, which provides high specificity, high yields and is ideally suited for applications that require sequence accuracy. Unlike other common proofreading enzymes, this polymerase is compatible with dUTP.

Yes. Reaction setup with SuperScript IV UniPrime One-Step RT-PCR System can be performed at room temperature. Moreover, due to the innovative two-phase hot start mechanism, preassembled reactions remain stable at room temperature for up to 24 hours for targets up to 3 kb and up to 4 hours for targets >3 kb.