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.

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.

Super efficient	Super sensitive	Super robust	Super fast	Super stable
Up to 100x higher cDNA yield	Ct values reduced by as many as eight cycles for RT-qPCR	Transcribes even from degraded or inhibitor-containing RNA samples	Ten-minute reaction times	High thermostability to transcribe structured templates

 Read white paper to see data

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 C_t values (ΔC_t = C_t – C_t SuperScript IV) show that SuperScript IV RT delivered up to 100x higher cDNA yields and has lower C_t values compared to SuperScript III and other commercial reverse transcriptases.

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For reliable cDNA synthesis, reverse transcriptases must offer high reaction sensitivity and low variability. Compared to other RTs, SuperScript IV Reverse Transcriptase has C_t 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 C_t values (reduced by 8 cycles). Moreover, it exhibits the lowest variation in RT-qPCR results to deliver reproducibility and the highest confidence results.

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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

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.

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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.

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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).

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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

Stand-alone enzymes for maximum flexibility in reaction setup:

•  SuperScript IV Reverse Transcriptase manual
•  SuperScript IV First-Strand Synthesis System manual
•  SuperScript IV VILO Master Mix manual
•  SuperScript IV UniPrime One-Step RT-PCR System manual
•  SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit manual
•  SuperScript IV CellsDirect cDNA Synthesis Kit manual

•  SuperScript IV reverse transcription protocols using SARS-CoV-2 research samples

•  White paper: SuperScript IV Reverse Transcriptase performance comparison to AMV-based enzymes
•  White paper: SuperScript IV Reverse Transcriptase
•  White paper: SuperScript IV VILO Master Mix
•  White paper: Efficient global preamplification via the SuperScript IV Single-Cell/Low-Input cDNA PreAmp Kit
•  Application note: Improved RT-qPCR analysis of difficult plant RNA samples
•  Application note: Reliable and sensitive RT-qPCR analysis of whole-blood RNA samples
•  Application note: Direct cDNA synthesis from single spheroids and primary cell samples
•  Poster: SuperScript IV Reverse Transcriptase

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