Up to 100-fold higher yield with degraded samples

Introducing the most robust reverse transcriptase for RT-PCR and RT-qPCR. Why settle for second best, when there are top IV reasons to love Invitrogen™ SuperScript™ IV reverse transcriptase.

  • Super efficiency – up to 100x higher cDNA yield
    from a wide range of sample types
  • Super fast – 10 minutes reaction time
  • Super sensitivity – reduced Ct values by as much as 8 for RT-qPCR
  • And it works  – resistance into a variety of reaction inhibitors

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SuperScript IV Reverse Transcriptase is available as stand-alone enzyme or in a first-strand synthesis system format. The first-strand synthesis system includes dNTPs, oligo(dT)20, random hexamers, control primers, and required buffer components. All necessary components are in separate tubes for maximum optimization of cDNA synthesis and to enable success for every experiment.


High efficiency reverse transcription

The ideal RT will reverse-transcribe even the most difficult types of RNA, such as RNA samples from plants (which are normally degraded, resulting in fewer copies of RNA transcripts).  SuperScript IV RT is so robust and efficient it can deliver up to 100x higher cDNA yield with degraded RNA as compared to other commercially available RTs (Figure 1).

Figure 1. Higher reverse transcription efficiency with degraded RNA using SuperScript IV RT. (A) A 1.2% Invitrogen™ E-Gel™ agarose gel with 500 ng total RNA from different samples. High-quality RNA (RIN >8) with intact ribosomal RNA is shown. Other RNA samples had a RIN score between 1 and 3 as determined on an Agilent Bioanalyzer instrument. (B) RTqPCR of degraded total RNA. Degraded RNA (100 ng) was used in a 20 μL SuperScript IV RT reaction with random hexamers according to the provided protocol. Reverse transcriptases from other vendors were used according to the manufacturers’ recommended protocols. From each RT reaction, 10% of the cDNA was added to Invitrogen™ EXPRESS qPCR Supermix, Universal (Cat. No. 1178501K), along with Applied Biosystems™ TaqMan™ Assays targeting the EF1A and ETIF5A genes. For each target, Ct values were normalized to Ct values obtained from the SuperScript IV RT sample.


RT Robustness in presence of reaction inhibitors

Inhibitory components typically found in RNA samples can interfere with cDNA synthesis and give false-negative RT-qPCR results. Such inhibitors can include reagents used during RNA extraction or co-purified components arising from biological samples, such as bile salts, SDS, or humic acid (Table 1). Compared with Invitrogen™ SuperScript™ III Reverse Transcriptase and reverse transcriptase enzymes from other vendors, SuperScript IV Reverse Transcriptase shows significant resistance to contaminating inhibitors (Figure 2). This helps ensure that you get good results, even with lower-purity RNA samples such as RNA with formalin and paraffin.

Table 1. Common cDNA synthesis inhibitors and their sources.

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

Figure 2. Analysis of first-strand cDNA synthesis performance in the presence of both biological and sample prep inhibitors. 500 ng 0.5–10 Kb RNA Ladder was used in a 10 µL SuperScript IV reverse transcriptase reaction with oligo(dT)20 according to provided protocol. Reverse transcriptase enzymes from other vendors were used according to the manufacturers’ recommended protocols. Inhibitors were added to total RNA prior to annealing to primer or addition of RT reaction mix. First-strand cDNAs were resolved by alkaline gel electrophoresis, and cDNA was stained using Applied Biosystems™ SYBR™ Gold Nucleic Acid Gel Stain. NaOH hydrolyzes all RNA, resulting in only visualization of cDNA.

 

Sensitive and reproducible cDNA synthesis with difficult samples

A thorough investigation of RT sensitivity and variability was performed for two Arabidopsis targets. Triplicate qPCR reactions were performed for each Input RNA amounts, e.g. 1, 10 and 100 ng (Figure 3). For both targets, SuperScript IV RT has the lowest Ct values over all input RNA amount with reduction by as much as 8. A plot of standard deviation for all input RNA amounts revealed that SuperScript IV RT has the lowest standard deviation with least amount of variation as compared to other commercially available RTs. 

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Figure 3. Sensitive and reproducible cDNA synthesis and low variability levels with a wide range of degraded plant RNA. 1–100 ng of degraded Arabidopsis total RNA (RIN: 1–3) was used in a 20 µL SuperScript IV reverse transcription reaction with random hexamers according to provided protocol. Reverse transcriptase enzymes from other vendors were used according to the manufacturers’ recommended protocols. 10% of cDNA in the RT reaction was added to TaqMan Assays for targets indicated in graph titles. Averaged Ct values were plotted against log10[input RNA in ng]. Standard deviation for each input RNA was plotted against RTs being tested.

RT reactions: n = 3 for each input RNA amount and RT qPCR: n = 3 for each RT reaction.


High thermostability for GC-rich templates and durability during the reaction

RNA secondary stem-and-loop structures and GC-rich RNA templates can interfere with cDNA synthesis. SuperScript IV Reverse Transcriptase exhibits high thermostability, which increases its ability to perform at higher temperatures (up to 55oC), helping to ensure successful transcription of RNA with high levels of secondary structure (Figure 4). SuperScript IV Reverse Transcriptase maintains its full activity throughout the RT reaction, resulting in better representation of full-length cDNA.

SuperScript IV Thermostability

Figure 4. High thermostability of SuperScript IV Reverse Transcriptase. 500 ng 0.5–10 Kb RNA Ladder was used in a 10 µL SuperScript reverse transcriptase reaction with oligo(dT)20 according to provided protocol, with the exception that reaction temperature was varied from 50–65°C. First-strand cDNAs were resolved by alkaline gel electrophoresis and cDNA was stained using SYBR Gold Nucleic Acid Gel Stain. NaOH hydrolyzes all RNA, resulting in only visualization of cDNA. Each cDNA band was measured by TotalLab software, and volumes were summed for each reaction temperature. Percent activity was calculated by taking the ratio of total volume at each reaction temperature to the total volume at 50°C.


Accelerated cDNA synthesis in 10 minutes with high yields of cDNA

SuperScript IV Reverse Transcriptase can synthesize a 9 kb cDNA in just 10 minutes, while SuperScript III Reverse Transcriptase (and competitors) cannot (Figures 5, 6).

Figure 5. Fast cDNA synthesis rate. Invitrogen™ 500 ng Millennium™ RNA Markers was used in a 10 µL SuperScript Reverse Transcriptase IV reaction with oligo(dT)20 according to provided protocol. Alternative reverse transcriptases were used according to the manufacturers’ recommended protocols. First-strand cDNAs were resolved by alkaline gel electrophoresis, and cDNA was stained using SYBR Gold Nucleic Acid Gel Stain. NaOH hydrolyzes all RNA, resulting in only visualization of cDNA.

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Figure 6. The cDNA synthesis protocol for SuperScript IV Reverse Transcriptase was developed to be easy to use while delivering full-length cDNA.

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