SuperScript™ IV Reverse Transcriptase, 10,000 Units - FAQs

Yes, you can use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase.

We have not tested this for SuperScript reverse transcriptases, so we cannot guarantee it would also work with those products.

This article can be used as a reference for additional information.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). 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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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).

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

Yes, SuperScript IV RT has TdT activity.

You can store the cDNA at -20 degrees C for up to 1 week. For long-term storage, we recommend storing the cDNA at -70 degrees C.

Regarding the tailing/TdT activity, a recent comparative analysis by R&D has shown that SuperScript IV RT has a much better TdT activity than SuperScript III RT, and that the latter has lost almost all activity.

Regarding the length of fragments that can be synthesized, internal tests have shown that fragments of up to 13 kb can be synthesized using SuperScript IV RT. Larger fragments have not been tested yet. Maxima H- RT was successfully tested with fragments up to 20 kb and since it has a certain amount of TdT activity, this enzyme could also be an alternative.
For template switching, we would recommend using SuperScript IV RT in the first instance, but Maxima H- RT would also work.

Yes, DNase treatment is highly recommended when any amplifcation will be performed after cDNA synthesis. Our ezDNase enzyme protocol is an extremely simplified genomic DNA removal step that occurs immediately before reverse transcription, and does not require re-purification of the RNA. This dramatically reduces the time of the entire reverse transcription protocol and reduces RNA loss or damage that can occur during conventional DNase treatment.

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 yield. This master mix formulation allows for a simpler reaction setup with less pipetting and less variation between samples.

SuperScript IV RT sustains 100% activity up to 56.4 degrees C and 70% activity up to 65 degrees C. The enzyme's ability to function at higher temperatures enables the reverse transcription of RNA targets with strong secondary structure.

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

Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

SuperScript IV RT functions exceedingly better than all tested enzymes in the presence of TRIzol Reagent, ethanol, SoluLyse reagent, guanidinium salts, LiCl, ammonium acetate, hematin, and humic acid. SuperScript IV RT is also able to perform in the presence of formalin, paraffin, and isopropanol.

We do not recommend using SuperScript III RT/ SuperScript II RT 5X first-strand buffer with Superscript IV RT. For optimal performance of Superscript IV RT, we recommend using it with the 5X RT buffer supplied in the kit.

The composition of the buffer is proprietary.

While retaining all the performance benefits of SuperScript III RT (Cat. No. 18080093), SuperScript IV RT (Cat. No. 18090010) has the following additional benefits:

- Significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis
- Robust and specific cDNA synthesis across a wide range of sample types
- Increased reproducibility
- A faster reverse transcriptase reaction time that reduces the incubation time from >50 minutes to 10 minutes
- Increased thermostability
- Significantly better processivity