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.
|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|
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.
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.
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, detergents||Sample preparation|
|Hematin, bile salts||Blood, feces|
|Humic acid, polyphenols, polysaccharides||Soil, plants|
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 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.
Figure 4. Fast cDNA synthesis capability. The Invitrogen Millennium RNA Marker was used in a 10 μL reaction with SuperScript IV Reverse Transcriptase and oligo(dT) primer according to the product protocol. Other commercially available RTs were used according to manufacturers’ protocols, except for reaction times reduced to 10 minutes. 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 visualization of cDNA only.
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 RT is available in several formats, including:
|SuperScript IV Reverse Transcriptase||SuperScript IV VILO Master Mix||SuperScript IV First-Strand Synthesis System||SuperScript IV One-Step RT-PCR System||SuperScript IV Single Cell/Low-Input cDNA PreAmp Kit||SuperScript IV CellsDirect cDNA Synthesis Kit|
|Applications||Most effective enzyme for all types of RNA, including difficult templates||First-strand cDNA synthesis reaction mix for two step RT-qPCR||Flexibility in reaction conditions||Fast and simple RT-PCR workflow||cDNA synthesis and preamplification from single cells or low quantities of RNA||Direct cDNA synthesis from mammalian cells|
|Optimal reaction temperature||50–55°C||50°C||50–55°C||50–55°C||50°C||50°C|
|RT reaction time||10 min||10 min||10 min||10 min||10 min||10 min|
Stand-alone enzymes for maximum flexibility in reaction setup:
|M-MLV-Reverse Transcriptase||SuperScript II Reverse Transcriptase||Superscript III Reverse Transcriptase||Superscript IV Reverse Transcriptase|
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 temperature||37°C||42°C||50°C||50–55°C|
|Reaction time||50 min||50 min||30–50 min||10 min|
|Sensitivity||1 ng||1 ng||10 pg||10 pg|
|Max cDNA length||Up to 7 kb||Up to 12.3 kb||Up to 12.3 kb||>12 kb|
|Ability to work with degraded or inhibitor-containing RNA||Low||Low||Medium||High|
|Reduced RNase H activity||No||Yes||Yes||Yes|
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.
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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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