Invitrogen™ SuperScript™ IV Reverse Transcriptase

Product Code.	No. of Reactions	unitSize
15307696	50 Reactions	10000 units
15387686	10 Reactions	2000 units
15317696	200 Reactions	40000 units

Product Code. 15307696 Supplier Invitrogen™ Supplier No. 18090050

Description

SuperScript IV Reverse Transcriptase (RT) is a proprietary MMLV mutant with superior robustness and reliability in RT reactions.

SuperScript IV Reverse Transcriptase (RT) is a proprietary MMLV mutant with superior robustness and reliability in RT reactions. It is significantly improved over SuperScript III RT in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of that enzyme, including increased thermostability, highly efficient full-length cDNA synthesis, and reduced RNase activity. SuperScript IV RT is designed to provide reliable, consistent, and fast cDNA synthesis in the presence of inhibitors found in a wide variety of samples that cause other currently available RTs to perform inefficiently.

SuperScript IV RT offers advantages at standard as well as elevated temperatures over cloned AMV and ThermoScript RT, including higher sensitivity, inhibitor tolerance, and ability to work with degraded RNA.

Features of SuperScript IV Reverse Transcriptase include:
• Significantly improved resistance to inhibitors that can interfere with cDNA synthesis
• Robust and specific cDNA synthesis in a wide range of sample types
• Faster reverse transcriptase reaction reduces incubation time from >50 minutes to 10 minutes
• Better processivity when compared to SuperScript III RT

SuperScript IV RT is the top choice for all RT-PCR and qRT-PCR applications in the SuperScript product family when reproducibility and reliability is the primary concern. It is also our top performing RT when inhibitors in the RNA sample may interfere with cDNA synthesis, leading to biases in gene expression studies.

Source
Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability and half-life, processivity, inhibitor resistance and to reduce RNase H activity

Unit definition
One unit of SuperScript IV RT is the amount of enzyme required to incorporate 1 nmole of deoxyribonucleotide into acid-precipitable material in 10 min at 37°C using poly(A) oligo(dT)_12-18 as a template/primer.

Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 4 mM MgCl₂, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [³H]-dTTP, 0.4 mM poly(A) oligo (dT)_12-18 and enzyme in 20 μl for 10 min at 37°C.

Specifications

• Concentration: 200 U/μL
• Content And Storage:

  • SuperScript IV RT, 50 μL (200 U/μL)
  • 5X RT buffer, 1 mL
  • 0.1 M DTT, 500 μL
  
  Store at -15°C to –25°C.
  
  

• Format: Stand-alone Enzyme
• GC-Rich PCR Performance: High
• Reaction Speed: 10 min.
• Technique: Reverse Transcription
• Optimal Reaction Temperature: 50°C
• Quantity: 10,000 Units
• Reverse Transcriptase: SuperScript IV
• Ribonuclease H Activity: Reduced
• Shipping Condition: Dry Ice
• Final Product Type: First-Strand cDNA
• No. of Reactions: 50 Reactions
• Reaction Format: Separate components
• Reagent Type: Reverse Transcription
• Size (Final Product): Up to 12.3 kb
• Starting Material: RNA

Frequently Asked Questions (FAQs)

Can I use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase (Cat. No. 28025013, 28025021)? Can other reverse transcriptases, such as SuperScript reverse transcriptase, be used in the same way?

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.

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

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 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

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.

Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.

Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

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).

Does SuperScript IV RT exhibit TdT activity?

Yes, SuperScript IV RT has TdT activity.

How long can I store the cDNA generated from my reverse transcription (RT) step?

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.

Do your 'engineered' MMLV-derived reverse transcriptase enzymes (e.g., SuperScript IV RT and Maxima RT) retain the ability to add a few additional nucleotides to the 3' end of the newly synthesized cDNA strand?

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.

Do I need to DNase treat my RNA when using SuperScript IV RT?

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.

What is the difference between SuperScript IV RT and the RT enzyme used in SuperScript IV VILO Master Mix?

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.

Can Superscript IV RT be used at higher reaction temperatures to reverse transcribe targets with secondary structure?

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.

What is the longest cDNA product that Superscript IV RT can synthesize?

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.

For more information about the performance of SuperScript IV, please refer to the whitepaper Whitepaper: SuperScript IV Reverse Transcriptase.

Which inhibitors has Superscript IV RT been tested with for superior performance?

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.

For more information about the performance of SuperScript IV, please refer to the white paper Whitepaper: SuperScript IV Reverse Transcriptase.

Can I use the Invitrogen SuperScript III RT/ Invitrogen SuperScript II RT 5X first-strand buffer with Invitrogen Superscript IV RT?

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.

What is the composition of the 5X RT buffer supplied with Invitrogen SuperScript IV RT?

The composition of the buffer is proprietary.

What are the unique features of Invitrogen Superscript IV Reverse Transcriptase? How does it compare to Invitrogen Superscript III Reverse Transcriptase?

While retaining all the performance benefits of SuperScript III RT, SuperScript IV RT 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

Safety and Handling

Product Identifier

• SuperScript IV Reverse Transcriptase

Signal Word

• Warning

Hazard Category

• Serious eye damage/eye irritation Category 2

Hazard Statement

• H319-Causes serious eye irritation.

Precautionary Statement

• P264-Wash hands thoroughly after handling.
• P280-Wear protective gloves/protective clothing/eye protection/face protection.
• P305+P351+P338-IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
• P337+P313-If eye irritation persists: Get medical advice/attention.

Supplemental information

• MIXTURE LIST-Contain: (R*,R*)-1,4-dimercapto-2,3-Butanediol

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