Figure 1. Amplification of long targets with high specificity in dramatically shorter time. Detection of 7.8 kb target from total HeLa RNA using SuperScript IV One-Step RT-PCR System, Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity, and supplier QI, N, QU, T, R, and BL one-step RT-PCR products. Reactions were performed according to supplier’s recommendations. Total one-step RT-PCR reaction times are indicated in hours:minutes. RNA target failed to amplify with products from suppliers QI and BL.
SuperScript IV One-Step RT-PCR System
Enabling faster, better RT-PCR
Even with challenging RNA samples, you can get better results faster and easier than with any other one-step RT-PCR reagent. The Invitrogen SuperScript IV One-Step RT-PCR System combines high-processivity Invitrogen SuperScript IV Reverse Transcriptase (RT) and high-fidelity Invitrogen Platinum SuperFi DNA Polymerase to provide superior one-step RT-PCR performance.
- Two-phase hot-start activation mechanism for high specificity, improved yields, and easy room-temperature setup
- Unmatched performance with sensitivity down to 0.01 pg of RNA, target length up to 13.8 kb, and the fastest one-step RT-PCR protocol
- Reliable target detection even in RNA samples with suboptimal purity
- Fast and easy gDNA removal for superior accuracy and confidence in your results
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Advantages of the SuperScript IV One-Step RT-PCR System
With the innovative two-phase, hot-start activation mechanism, the SuperScript IV One-Step RT-PCR System generates specific product at high yields in half the reaction time compared to other commercial kits.
The high sensitivity of SuperScript IV One-Step RT-PCR System enables detection of low-abundance RNA targets. High sensitivity is advantageous in experiments where there is a limited amount of starting material or the concentration of target RNA in the sample is low.
Figure 2. High sensitivity and reliable target detection from low amounts of input RNA. Amplification of 0.35 kb fragment from 0.01 pg, 1 pg, 10 pg, 10 ng, and 100 ng total HeLa RNA with the SuperScript IV One-Step RT-PCR System, SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity, and supplier QI, N, QU, T, BL, and R one-step RT-PCR products.
Due to the high processivity of SuperScript IV RT and Platinum SuperFi DNA Polymerase, the SuperScript IV One-Step RT-PCR System enables detection of a broad range of target lengths.
The SuperScript IV One-Step RT-PCR System is able to withstand the effect of common RT and PCR inhibitors such as copurified compounds from biological samples or reagents used for RNA purification. This exceptionally robust system minimizes dependency on RNA-sample purity for reliable results.
Figure 4. Resistance to inhibitors. Detection of a 1 kb RNA target from total HeLa RNA using the SuperScript IV One-Step RT-PCR System, SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity, and supplier QI, N, QU, T, BL, and R one-step RT-PCR products in reaction mixtures containing:
2–Heparin (0.18 µg/uL)
3–Xylan (2.5 µg/uL)
4–Humic acid (0.02 µg/uL
5–LiCl (2 µg/uL).
All kits except the SuperScript IV One-Step RT-PCR System were inhibited with indicated amounts of inhibitors.
Stability of SuperScript IV One-Step RT-PCR reactions at room temperature for an extended time enables high-throughput applications.
- RNA purification methods, including protocols with DNase digestion on-column, often fail to remove gDNA completely. Amplification of contaminating gDNA can cause non-specific or misleading one-step RT-PCR results.
- Traditional gDNA decontamination protocols with DNase I include time consuming DNase inactivation or removal steps under conditions that can damage RNA and affect results.
- The new SuperScript IV One-Step RT-PCR System with ezDNase Enzyme allows efficient, fast, and gentle (5 min at 37°C) gDNA elimination from RNA samples and ensures the highest accuracy and confidence in one-step RT-PCR results.
With its high robustness SuperScript IV One-Step RT-PCR system can amplify several targets in the same multiplex one-step RT-PCR reaction with high efficiency and specificity.
Figure 6. Simultaneous amplification of up to 4 targets using SuperScript IV One-Step RT-PCR System. One or more targets of increasing sizes (528, 997, 3,009, and 4,497) was amplified using total HeLa RNA as template.
Frequently asked questions
The optimal annealing temperature for your primers may differ significantly when using the SuperScript IV One-Step RT-PCR System in comparison to other one-step RT-PCR products due to differences in buffer salt concentration. Always use the Tm calculator (thermofisher.com/tmcalculator) to determine your primers’ Tm values and recommended annealing temperature.
The RT-PCR cycling conditions with the SuperScript IV One-Step RT-PCR System differ significantly from other one-step RT-PCR products. For the best results, always use cycling conditions described in the SuperScript IV One-Step RT-PCR System manual.
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 One-Step RT-PCR system, cDNA synthesis can be performed at higher temperatures than with 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 55–60 °C.
The two-phase hot-start mechanism ensures sequential activation of RT and PCR enzymes in the one-step RT-PCR workflow. At ambient temperature, SuperScript IV RT is maintained 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 Platinum SuperFi 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.
Platinum SuperFi DNA Polymerase in the SuperScript IV 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.
Annealing temperature rules for Platinum SuperFi DNA Polymerase are different from many common DNA polymerases (such as Taq DNA polymerase). For optimal one-step RT-PCR results with SuperScript IV One-Step RT-PCR System, use the Tm calculator on our website.
For Research Use Only. Not for use in diagnostic procedures.