What is one-step RT-PCR?

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.

Learn about the differences between one-step and two-step RT-PCR

RT selection tool screenshot


Invitrogen One-Step RT-PCR Systems Selection Guide


Table 1: Comparison of one-step RT-PCR systems

 SuperScript IV UniPrime One-Step RT-PCR SystemSuperScript IV One-Step RT-PCR SystemSuperScript III One-Step RT-PCR System with Platinum Taq DNA PolymeraseSuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase
Sensitivity0.01 pg0.01 pg0.01 pg1 pg
Range of amplicon length13 kb13.8 kb4.5 kb10 kb
Benchtop stability at RT24 h for up to 3 kb
4 h for ≥3 kb
Up to 4 h
Resistance to inhibitors*******
Multiplex*******
RT time10 min10 min30 min30 min
Extension time30 s/kb30 s/kb1 min/kb1 min/kb
Universal annealingYesNoNoNo
Tracking dyeYesNoNoNo
Integrated gDNA removalNoYesNoNo

*Indicates degree of resistance to inhibitors and ability to multiplex
A separate format with Invitrogen ezDNase Enzyme


Advantages of the SuperScript IV UniPrime One-Step RT-PCR System

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 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)

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).

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).

Resources for SuperScript IV UniPrime One-Step RT-PCR System

Learn how the SuperScript IV UniPrime One-Step RT-PCR System makes it easy to set up reactions while bringing superior results.

OEM one-step RT-PCR

To manufacture products according to your specifications, leverage our industry-leading expertise and infrastructure as a comprehensive third-party OEM service. We provide kitting, labeling, and commercialization services for one-step RT-PCR. As a vertically integrated organization, we control reagent quality throughout the nucleic acid synthesis process which reduces batch-to-batch variability.

To inquire about OEM one-step RT-PCR and other enzymes: Contact us

Learn more: OEM & commercial supply services


One-Step RT-PCR Systems: Frequently asked questions

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

Resources

Reverse transcription technical resources

Learn about the reverse transcription basics, setup considerations, importance of enzyme polymerase choice, common methods and applications, and troubleshooting.

For Research Use Only. Not for use in diagnostic procedures.

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