What is SuperScript IV Reverse Transcriptase?

SuperScript IV Reverse Transcriptase (RT) is the latest generation RT from Invitrogen SuperScript product family. It is a proprietary MMLV mutant with reduced RNase H activity, increased thermostability, and highly efficient full-length cDNA synthesis. Compared to previous generation SuperScript RTs, SuperScript IV reverse transcriptase has significantly improved processivity, which enables fast reaction speed, inhibitor resistance, and exceptional performance even with challenging RNA samples. SuperScript IV reverse transcriptase is available in multiple formats tailored to specific applications, and it is widely cited in peer reviewed journals.

Advantages of SuperScript IV Reverse Transcriptase

SuperScript IV Reverse Transcriptase is known for its efficiency, sensitivity, robustness, short-reaction time, and thermostability. Click on the attributes below to see supporting data. For recommendations on choosing the reverse transcriptase fit for your application, use the selection tool.

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SuperScript IV Reverse Transcriptase: efficiency

An efficient RT will transcribe low abundance RNA or degraded RNA. SuperScript IV RT is very robust and efficient and can deliver up to 100x higher cDNA yields with degraded RNA than other commercially available RTs (Figure 1). SuperScript IV RT is a exceptional choice for cDNA synthesis with any type of RNA and represents a unique solution for degraded or limited RNA.

Figure 1. High efficiency with degraded RNA. RT-qPCR of degraded RNA (RIN 1–3) from human cells and plant tissues was performed with different brands of commercially available RTs and Applied Biosystems TaqMan assays. Delta Ct values (ΔCt = Ct – Ct SuperScript IV) show that SuperScript IV RT delivered up to 100x higher cDNA yields and has lower Ct values compared to SuperScript III and other commercial reverse transcriptases.

SuperScript IV Reverse Transcriptase: sensitivity

For reliable cDNA synthesis, reverse transcriptases must offer high reaction sensitivity and low variability. Compared to other RTs, SuperScript IV Reverse Transcriptase has Ct values reduced by 8 cycles (Figure 2). In triplicate RT-qPCR reactions performed with three different amounts of degraded RNA input, SuperScript IV Reverse Transcriptase had the lowest Ct values (reduced by 8 cycles). Moreover, it exhibits the lowest variation in RT-qPCR results to deliver reproducibility and the highest confidence results.

Figure 2. Sensitivity and variability in cDNA synthesis using degraded RNA samples. Degraded Arabidopsis total RNA (RIN: 1–3), in amounts of 1, 10, and 100 ng were used as input RNA in 20 μL reverse transcription reactions with SuperScript IV Reverse Transcriptase and random hexamers according to the product protocol. RTs from other vendors were used according to the manufacturers’ protocols. For each tested enzyme and each RNA input, RT reactions were performed in triplicates. From each reverse transcription reaction, 10% of the cDNA product was added to TaqMan assays for two targets, Gln synthetase and WRKY TF 70. Three qPCR reactions were performed for each reverse transcription reaction and the average Ct values for each RNA input were plotted (standard deviation from 9 Ct values for each input RNA).

SuperScript IV Reverse Transcriptase: inhibitor tolerance

Compounds that have inhibitory effects on RTs are commonly found in RNA samples even after thorough purification. These compounds may interfere with cDNA synthesis, produce false RT-PCR and RT-qPCR results, and cause results to be misinterpreted. RT inhibitors include reagents used during RNA extraction, and co-purified contaminants arising from biological samples (Table 1). SuperScript IV Reverse Transcriptase shows significantly improved resistance to contaminating inhibitors when compared to previous generation SuperScript III Reverse Transcriptase and other commercially available RTs (Figure 3).

Table 1. Common cDNA synthesis inhibitors and their sources

Ethanol/isopropanol, salts, phenol/chloroform, detergentsSample preparation
Hematin, bile saltsBlood, feces
Humic acid, polyphenols, polysaccharides Soil, plants
Formalin, paraffinFFPE

Figure 3. Higher performance in cDNA synthesis in the presence of biological or sample prep inhibitors. A 0.5–10 kb RNA ladder was used in a 10 μL SuperScript IV Reverse Transcriptase reaction with oligo(dT)20 according to the product protocol. RTs from other vendors were used according to their respective protocols. Inhibitors were added to the RNA samples prior to primer annealing or addition of RT reaction mix. First-strand cDNAs were resolved by alkaline gel electrophoresis, and cDNA was stained using Invitrogen SYBR Gold Nucleic Acid Gel Stain. During electrophoresis, NaOH hydrolyzes all RNA, resulting in visualization of cDNA only.

SuperScript IV Reverse Transcriptase: reaction time

SuperScript IV Reverse Transcriptase has remarkable processivity and thereby generates long, full-length cDNA fragments within a short reaction time. Figure 4 demonstrates that SuperScript IV Reverse Transcriptase synthesized cDNAs of up to 9 kb in 10 minutes, while most other commercially available RTs were only able to synthesize cDNAs between 1.5–3 kb or less in the same duration.

SuperScript IV Reverse Transcriptase: thermostability

When reverse transcription reactions are performed at low temperatures (less than 42°C), RNA secondary structures, especially GC-rich templates, may interfere with cDNA synthesis. SuperScript IV Reverse Transcriptase has high thermostability and can be used in reactions at 50°C or higher, which facilitates the successful transcription of highly structured RNA transcripts (Figure 5).

Figure 5. High thermostability of SuperScript IV Reverse Transcriptase. A 0.5–10 kb RNA ladder was used in a 10 μL SuperScript IV RT reaction with oligo(dT) according to the product protocol, with the exception that reaction temperature was varied between 50 and 65°C. First-strand cDNAs were resolved by alkaline gel electrophoresis, and cDNA was stained using SYBR Gold Nucleic Acid Gel Stain. During electrophoresis NaOH hydrolyzes all RNA, resulting in the visualization of cDNA only. cDNA bands were quantitated by TotalLab software for each reaction temperature. Percentage SuperScript IV RT activity was calculated by dividing values at each reaction temperature by values at 50°C.

SuperScript IV Reverse Transcriptase formats: selection guide

SuperScript IV RT is available in several formats, including:

  • Stand-alone enzyme—reverse transcriptase supplied with reaction buffer
  • First-strand synthesis system—all cDNA synthesis reaction components are in separate tubes for maximum flexibility of reaction conditions
  • Master mix—cDNA synthesis reaction components are premixed for exceptional efficiency and reduced variability in RT-qPCR applications
  • One-step RT-PCR system—RT and PCR reagents for one-step RT-PCR applications
  • Direct reverse transcription—reagents for cell lysis and cDNA synthesis included; RNA purification is not required


 SuperScript IV Reverse Transcriptase
SuperScript IV VILO Master MixSuperScript IV First-Strand Synthesis SystemSuperScript IV One-Step RT-PCR System
SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit
SuperScript IV CellsDirect cDNA Synthesis Kit
ApplicationsMost effective enzyme for all types of RNA, including difficult templatesFirst-strand cDNA synthesis reaction mix for two step RT-qPCRFlexibility in reaction conditionsFast and simple RT-PCR workflowcDNA synthesis and preamplification from single cells or low quantities of RNADirect cDNA synthesis from mammalian cells
Optimal reaction temperature50–55°C50°C50–55°C50–55°C50°C50°C
RT reaction time10 min10 min10 min10 min10 min10 min
Available formats

Comparison of SuperScript IV Reverse Transcriptase with other RTs

Stand-alone enzymes for maximum flexibility in reaction setup:

 M-MLV-Reverse Transcriptase
SuperScript II Reverse TranscriptaseSuperscript III Reverse Transcriptase
Superscript IV Reverse Transcriptase
Key attribute

Recombinant M-MLV RT for routine, non-demanding applications

Engineered M-MLV RT with reduced RNase H activity

Engineered M-MLV RT with reduced RNase H activity and improved thermal stability

Latest generation engineered RT for exceptional cDNA synthesis performance

Optimal reaction temperature37°C42°C50°C50–55°C
Reaction time50 min50 min30–50 min10 min
Sensitivity1 ng1 ng10 pg10 pg
Max cDNA lengthUp to 7 kbUp to 12.3 kbUp to 12.3 kb>12 kb
Ability to work with degraded or inhibitor-containing RNALowLowMediumHigh
Reduced RNase H activityNoYesYesYes

Resources for SuperScript IV Reverse Transcriptase

Reverse transcriptases: Selection tool

Too many options and not sure which format to use, try the selection tool for recommendations.

Watch this video on the advantages of SuperScript IV Reverse Transcriptase.

Understand how SuperScript IV Reverse Transcriptase consistently delivers low Ct values in qPCR reactions.

Discover how SuperScript IV Reverse Transcriptase performs despite presence of inhibitors.



Technical articles

SuperScript IV Reverse Transcriptase FAQs

Find tips, troubleshooting help, and resources for common questions about SuperScript Reverse Transcriptases. Can’t find your question? Search the FAQ database

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.

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


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