SuperScript IV Reverse Transcriptase: Frequently Asked Questions

The SuperScript IV enzyme has been engineered for higher thermostability, processivity, and cDNA yields. It performs better in the presence of inhibitors, and the reaction buffer has also been optimized for robust cDNA synthesis from a wide range of samples.

When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density, below).

The only change is that the incubation time for the reverse transcription reaction has been reduced from 50 minutes to 10 minutes. All the other parameters and steps are the same.

While wild type MMLV RT or MMLV RNase H^– RT enzymes usually display very low or no activity above 45°C, SuperScript IV RT sustains 100% activity at up to 56.4°C and 70% activity at up to 65°C. SuperScript IV RT’s ability to function at higher temperatures enables the reverse transcription of RNA targets with structural complexities.

We have found that SuperScript IV RT was able to produce a 12.3 kb product, while all other RTs tested at the same time produced smears and smaller products, using a gene-specific reverse transcription primer.

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix. The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.

The SuperScript IV VILO Master Mix contains SuperScript IV RT, a ribonuclease inhibitor, and a helper protein. The helper protein helps to increase the efficiency of the reverse transcription reaction and thus improve cDNA production. This master mix formulation allows for a simpler reaction setup with less pipetting and less variation between samples.

SuperScript IV VILO Master Mix uses SuperScript IV RT in the optimized master mix formulation, whereas SuperScript VILO products are based on SuperScript III RT. Compared to SuperScript III RT, SuperScript IV RT has significantly improved performance due to higher thermostability, processivity, and the ability to synthesize cDNA efficiently from a wide variety of RNA samples, even those of suboptimal purity and integrity

The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50°C). The additional inactivation step is therefore not required in RT-qPCR applications.

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 T_m calculator (thermofisher.com/tmcalculator) to determine your primers’ T_m 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.