Superior cDNA synthesis with SuperScript IV Reverse Transcriptase

Superior cDNA synthesis with SuperScript IV Reverse Transcriptase

The Invitrogen SuperScript line of reverse transcriptases (RTs) represents the most widely used and highly referenced RTs for cDNA synthesis in many applications. Invitrogen Superscript IV RT, the most recently introduced enzyme, was engineered to offer superior cDNA synthesis performance with even the most challenging RNA samples.

Why settle for second best, when there are top IV reasons to love SuperScript IV RT:

  • 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

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Order SuperScript IV Reverse Transcriptase products

SuperScript IV RT is available as a stand-alone enzyme, in a first-strand synthesis system format, and in a master mix format. The first-strand synthesis system includes all necessary cDNA synthesis components in separate tubes for maximum optimization of reaction conditions. In the master mix format, all the cDNA synthesis reaction components are premixed for superior efficiency and reduced variability in RT-qPCR applications.

  SuperScript IV Reverse Transcriptase SuperScript IV First-Strand Synthesis System SuperScript IV VILO Master Mix
Format Stand-alone enzyme cDNA synthesis kit cDNA synthesis master mix for two step RT-qPCR
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SuperScript IV Reverse Transcriptase advantages

Efficient reverse transcription of low-abundance or degraded RNA

An efficient RT will reverse-transcribe even the most difficult types of RNA, including those that are of low abundance or degraded. 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 superior 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 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.

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 (Figure 2). 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.

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Figure 2. 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 (Table 1). SuperScript IV RT shows significantly better resistance to contaminating inhibitors than Invitrogen SuperScript III and other commercially available RTs (Figure 3).

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 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 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.

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 4 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.

 

Figure 4. 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.

Confidently transcribe structured templates

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 (Figure 5).

Figure 5. High thermostability of SuperScript IV RT. 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 visualization of cDNA only. cDNA bands were quantitated by TotalLab software for each reaction temperature. Percent of SuperScript IV RT activity was calculated by dividing values at each reaction temperature by values at 50°C.