One-step reverse transcription-polymerase chain reaction (RT-PCR) is one of the most common techniques for RNA analysis. During RT-PCR, RNA molecules are first converted into complementary DNA (cDNA), which is then amplified by PCR. In the one-step RT-PCR approach, the reverse transcriptase and the DNA polymerase are premixed into a single tube, allowing both the RT and the subsequent PCR step to be performed in a single reaction. The advantages of one-step RT-PCR over two-step RT-PCR include fast and simple analysis, less pipetting steps, lower risk of errors and contamination, and suitability for high-throughput applications.
Table 1: Comparison of one-step RT-PCR systems
|SuperScript IV UniPrime One-Step RT-PCR System||SuperScript IV One-Step RT-PCR System||SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase||SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase|
|Sensitivity||0.01 pg||0.01 pg||0.01 pg||1 pg|
|Range of amplicon length||13 kb||13.8 kb||4.5 kb||10 kb|
|Benchtop stability at RT||24 h for up to 3 kb|
4 h for ≥3 kb
|Up to 4 h||—||—|
|Resistance to inhibitors||***||**||*||*|
|RT time||10 min||10 min||30 min||30 min|
|Extension time||30 s/kb||30 s/kb||1 min/kb||1 min/kb|
|Integrated gDNA removal||No||Yes†||No||No|
The innovative technology used in the SuperScript IV UniPrime One-Step RT-PCR System does not require Tm 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 Tm 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.
Figure 4A. Extended stability at room temperature. Using the SuperScript IV UniPrime One-Step RT-PCR System, one-step RT-PCR reactions were assembled with 1 kb RNA target from 1 ng—1 pg UHRR. One batch of assembled reactions was immediately loaded to the thermal cycler. The second batch of assembled reactions was kept at room temperature for 24 hours, and then loaded to the thermal cycler. Analysis of RT-PCR products shows that specific target amplification was achieved even after extended time at room temperature. NTC: no-template control. The molecular weight marker is Thermo Scientific GeneRuler 1 kb Plus DNA Ladder, ready-to-use.
Figure 4B. Extended stability at room temperature. Using the SuperScript IV UniPrime One-Step RT-PCR System, one-step RT-PCR reactions were assembled with 4.5 kb RNA target from 1 ug—1 pg of UHRR. One batch of assembled reactions was immediately loaded to the thermal cycler. The second batch of assembled reactions was kept at room temperature for 4 hours, and then loaded to the thermal cycler. Analysis of RT-PCR products shows that specific target amplification was achieved even after extended time at room temperature. NTC: no-template control. The molecular weight marker is Thermo Scientific GeneRuler 1 kb Plus DNA Ladder, ready-to-use.
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.
With features such as high specificity, processivity, and universal annealing, the SuperScript IV UniPrime One-Step RT-PCR System can successfully multiplex without the need for significant optimization (Figure 6).
Figure 6A. Multiplex RT-PCR from various RNA input amounts. 10 targets (101, 131, 199, 301, 346, 399, 498, 613, 734, and 1006 bp) were amplified from 0.1–1000 ng of UHRR. The molecular weight marker is Thermo Scientific GeneRuler 100 bp Plus DNA Ladder, ready-to-use.
Figure 6B. Multiplex RT-PCR across broad range of target lengths. 7-plex (0.2 kb, 0.5 kb, 1kb, 1.5 kb, 3 kb, 5.7 kb and 9.4 kb) and 6-plex (0.2 kb, 0.4 kb, 0.7 kb, 1 kb, 1.5 kb, and 3 kb) RT-PCR was performed from 100–1000 ng of UHRR. The molecular weight marker is Thermo Scientific GeneRuler 1 kb Plus DNA Ladder, ready-to-use.
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).
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.
For Research Use Only. Not for use in diagnostic procedures.
© 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.