Thermo Fisher Scientific

Invitrogen™ SuperScript™ IV Reverse Transcriptase DNA Polymerase

Invitrogen™ 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 in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous enzyme, including increased thermostability, highly efficient full-length cDNA synthesis, and reduced RNase activity. Thermo Fisher Scientific™ Invitrogen™ 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 Reverse Transcriptase

  • Super-efficient–up to 100x higher cDNA yield
  • Super-sensitive–Ct values reduced by as many as 8 cycles for RT-qPCR
  • Super-robust–transcribes even from degraded or inhibitor-containing RNA samples
  • Super-fast–10 min reaction time

Increased Sensitivity and Reproducibility with Challenging RNA Samples

For the greatest reliability in cDNA-based experiments, RTs should offer high reaction sensitivity and low variability. For example, triplicate RT-qPCR reactions were performed with three different amounts of degraded RNA input and different RTs. The results demonstrate that SuperScript IV RT resulted in the lowest Ct values (reduced by as much as 8 cycles) and the lowest standard deviations when compared to other commercially available RTs. This confirms that SuperScript IV RT offers superior reaction sensitivity and reproducibility to deliver the highest-confidence results.

 
High efficiency with degraded RNA. RT-qPCR of degraded RNA (RIN 1–3) from human cells and plant tissues was performed with different 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 lower Ct values than SuperScript III and other RTs.





















SuperScript™ IV RT is the best choice for all RT-PCR and qRT-PCR applications in the SuperScript™ product family. Particularly when reproducibility and reliability are primary concerns and where inhibitors in the RNA sample can interfere with cDNA synthesis and cause bias in gene expression studies, the Invitrogen™ line offers unique solutions.

Features of SuperScript™ IV Reverse Transcript include:

  • Significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis
  • Robust and specific cDNA synthesis in a wide range of sample types
  • A faster reverse transcriptase reaction that reduces the incubation time from >50 minutes to 10 minutes
  • Significantly better processivity compared to SuperScript® III RT

 

Increased Sensitivity and Reproducibility with Challenging RNA Samples

For the greatest reliability in cDNA-based experiments, RTs should offer high reaction sensitivity and low variability. For example, triplicate RT-qPCR reactions were performed with three different amounts of degraded RNA input and different RTs. The results demonstrate that SuperScript IV RT resulted in the lowest Ct values (reduced by as much as 8 cycles) and the lowest standard deviations when compared to other commercially available RTs. This confirms that SuperScript IV RT offers superior reaction sensitivity and reproducibility to deliver the highest-confidence results.






 
Sensitive and reproducible cDNA synthesis using challenging RNA samples. Degraded Arabidopsis total RNA (RIN: 1–3), in amounts of 1–100 ng, was used in 20 μL SuperScript IV RT reactions with random hexamers according to the product protocol. RTs from other vendors were used according to the manufacturers’ recommended protocols. For each RT enzyme, three reverse transcription reactions were performed for each input RNA. 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).
























 

Robust Transcription in the Presence of Inhibitors

Numerous compounds that have inhibitory effects on RTs are commonly found in RNA samples even after employing thorough purification methods. These compounds can interfere with cDNA synthesis, result in false RT-PCR and RT-qPCR results, and cause misinterpretation of the experimental system. RT inhibitors include reagents used during RNA extraction, and co-purified contaminants arising from biological samples. SuperScript IV RT shows significantly better resistance to contaminating inhibitors than Invitrogen SuperScript III and other commercially available RTs.
 

Common cDNA Synthesis Inhibitors and Their Sources

Inhibitor Source
Ethanol/isopropanol, salts, phenol/chloroform, detergents Sample prep
Hematin, bile salts Blood, feces
Humic acid, polyphenols, polysaccharides Soil, plants
Formalin, paraffin FFPE










 
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 RT reaction with oligo(dT)20 according to the product protocol. RTs from other vendors were used according to their respective recommended 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

cDNA Synthesis in Just 10 Minutes

SuperScript IV RT is a highly processive enzyme that can rapidly perform cDNA synthesis to generate long, full-length cDNA fragments. Figure demonstrates that SuperScript IV RT synthesized cDNAs of up to 9 kb in just 10 minutes, while most other commercially available RTs were only able to synthesize cDNAs between 1.5–3 kb or less in the same amount of time.
 
Fast cDNA synthesis capability. The Invitrogen Millennium RNA Marker was used in a 10 μL SuperScript IV RT reaction with oligo(dT) according to the product protocol. Alternative RTs were used according to their respective recommended protocols, except that reaction times were reduced to 10 min. 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.



















 


High Thermostability with Reverse Transcriptase

RNA secondary structures, especially with GC-rich templates, can interfere with cDNA synthesis, especially if the reaction is carried out at low temperatures (less than 42°C). SuperScript IV RT has high thermostability and can be used in reactions at temperatures of 50°C or higher, helping to ensure successful transcription of even highly structured RNA transcripts.
 

 
RNA secondary structures, especially with GC-rich templates, can interfere with cDNA synthesis particularly if the reaction is performed at low temperatures (less than 42°C). SuperScript IV RT has high thermostability and can be used in reactions at 50°C or higher, which helps to ensure successful transcription of even highly structured RNA transcripts.




















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.

Performance and Quality Testing

  • Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product.

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.

Content&Storage

  • SuperScript™ IV RT (2,000 units total at 200 U/µL), 10 µL
  • 5X RT buffer, 1 mL
  • 0.1 M DTT, 500 µL
  • Store at -5 to -30°C.

SuperScript™ IV Reverse Transcriptase Specifications

Optimal Reaction Temperature 50° C
Product Line SuperScript™
Reverse Transcriptase SuperScript™ IV
Ribonuclease H Activity Reduced
Shipping Condition Dry Ice
Quantity 2,000 units
Size (Final Product) 12.3 kb or less












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