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Invitrogen SuperScript IV Reverse Transcriptase established a strong foundation for fast, robust cDNA synthesis, with high processivity, thermostability, and inhibitor tolerance. For researchers using standard reverse transcription workflows, the next-generation SuperScript V family builds on these proven strengths with further advances in performance, stability, and workflow convenience.
SuperScript IV technology also powers a range of established specialty solutions designed for specific reverse transcription applications and workflows. Explore SuperScript IV solutions for one-step RT-PCR, single-cell and low-input cDNA preamplification, direct cDNA synthesis from cells, template switching, and other specialized applications, along with supporting protocols, application data, and technical resources.
SuperScript IV Reverse Transcriptases for specialty applications formats
- 2-step RT-qPCR
- One-step RT PCR
- Preamplification
- Template Switching
- cDNA from Cells
cDNA synthesis and preamplification from single cells or low quantities of RNA.
Full length cDNA synthesis with enhanced template switching efficiency. Available as kits and separate reagents.
Direct cDNA synthesis from mammalian cell lysates.
Performance of SuperScript IV VILO Master Mix
- Reliable
- Efficient
- Challenging RNA samples
- gDNA removal
- Fast workflow
Reliable cDNA synthesis for qPCR
SuperScript IV VILO Master Mix contains a proprietary helper protein which improves the interaction between SuperScript IV Reverse Transcriptase and the template RNA, and thereby, extends linearity across ten orders of magnitude for input RNA (Figure 1). Continue to get a high degree of linearity across a wide range of target inputs with our qPCR master mixes that are specifically formulated for highly sensitive and accurate gene expression analysis.
Figure 1. Linearity across 10 orders of magnitude for a range of RNA input. Serial dilutions of total RNA from HeLa cells were reverse transcribed using the SuperScript IV VILO Master Mix, followed by qPCR reactions using human TaqMan assay for 18S rRNA with the Invitrogen EXPRESS qPCR SuperMix Universal. Even across a wide range of RNA input from 1 fg to 1 μg, the master mix exhibits a coefficient of correlation of 0.999 and high efficiency of 94.2%. The amplification plot illustrates the robust nature of the SuperScript IV VILO Master Mix over a broad range of RNA input. This means that you can normalize your lower-abundance genes to your reference genes without worrying about potential variation of RT efficiency at different RNA input levels.
Efficient cDNA synthesis for qPCR
Higher cDNA synthesis efficiency of SuperScript IV VILO Master Mix (Figure 2) allows for using lower RNA inputs in RT-qPCR workflow, better RT-qPCR sensitivity to work with low-copy targets and degraded RNA, and archiving cDNA for future studies.
Figure 2. Highest efficiency across a broad range of targets. cDNA synthesis was performed with different master mixes per manufacturer instructions, using 1 ng of total HeLa RNA input. qPCR was performed with Invitrogen EXPRESS qPCR SuperMix and Applied Biosystems TaqMan primer and/or probes for indicated gene targets. Delta Ct values (∆Ct= Ct – CtSuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and on average two cycles lower Ct values compared to other RNA to cDNA synthesis kits.
cDNA synthesis with challenging RNA samples
Typically, cDNA synthesis for qPCR requires high quality intact RNA samples to achieve accurate RT-qPCR results. However, SuperScript IV VILO Master Mix shows robust performance even when challenged with difficult samples, containing common reaction inhibitors (Figure 3A) or degraded RNA (Figure 3B).
Figure 3A. Exceptional performance with inhibitor-containing RNA. cDNA synthesis was performed using 100 ng HeLa in reactions containing different inhibitors. qPCR was performed with TaqMan primer/probes for the B2M gene target using EXPRESS qPCR SuperMix. Delta Ct values (∆Ct= Ct – CtSuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered maximum cDNA yield and minimal Ct values in presence of all tested reaction inhibitors.
Figure 3B. Exceptional performance with degraded RNA. cDNA synthesis was performed using 50 ng of degraded (RIN<5) RNA from frozen lung tissue. qPCR was performed with TaqMan primer/probes for different gene targets using EXPRESS qPCR SuperMix. Delta Ct values (∆Ct= Ct – CtSuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield at lowest Ct values compared to other RNA to cDNA kits.
RNA-friendly gDNA removal in two minutes
RNA purification methods are not foolproof and often fail to completely remove gDNA. Amplification of contaminating gDNA can cause a shift in Ct values, especially when detecting poorly expressed genes. DNase I enzyme is commonly used to remove gDNA from RNA. However, DNase I may degrade single stranded DNA such as primers and cDNA, and therefore DNase I must be inactivated or removed prior to cDNA synthesis. DNase I removal processes using EDTA or other methods can damage or reduce yields of RNA (Figure 4A).
The gDNA removal step is simplified with the SuperScript IV VILO Master Mix format with ezDNase enzyme. The Invitrogen ezDNase enzyme exhibits double stranded DNA-specific activity and allows gDNA removal in two minutes at 37°C. The ezDNase enzyme is thermolabile, and is inactivated at 50°C, which is the standard SuperScript IV RT cDNA synthesis temperature. This attribute of ezDNase eliminates the need for a separate inactivation step and enables accuracy and confidence in RT-qPCR results (Figure 4A and 4B).
Figure 4A. Effect of gDNA removal with DNase I and ezDNase on Ct values. HeLa total RNA was treated with ezDNase or DNase I enzymes. Samples treated with ezDNase enzyme were immediately processed for RT-qPCR, while those treated with DNase I were first processed for DNase I inactivation in the presence of EDTA according to standard protocols. Both RNA samples were serially diluted into duplicate RT-qPCR reactions with SuperScript IV VILO Master Mix and TaqMan 18S rRNA assay. Treatment with DNase I resulted in later Ct values (by 0.5 cycles on average), suggesting that DNase I treatment and inactivation negatively affected RNA integrity and/or yields.
Figure 4B. gDNA decontamination with ezDNase enzyme. 100 ng of human gDNA was mixed with 250 ng HeLa total RNA. Three different reactions were performed with SuperScript IV VILO Master Mix and qPCR assays specific for the gDNA target: RT-qPCR, qPCR, and qPCR with sample treated with ezDNase for 2 minutes at 37°C. ezDNase effectively removed gDNA and resulted in no target amplification.
Simplified and fast workflow
Due to the high processivity of SuperScript IV RT in the SuperScript IV VILO Master Mix, cDNA synthesis reactions can be significantly faster (10 minutes) in comparison to reactions performed with traditional RT enzymes (60 minutes). Furthermore, the protocol for gDNA removal with ezDNase takes as little as two minutes and does not require a dedicated enzyme inactivation step. Therefore, the workflow for cDNA synthesis involving gDNA removal with the SuperScript IV VILO Master Mix can be significantly shorter than with traditional RNA to cDNA kits (Figure 5).
Figure 5. Workflow comparisons. Comparison between the SuperScript IV VILO Master Mix cDNA synthesis workflow including sample treatment with ezDNase enzyme (top) and the traditional cDNA synthesis workflow with DNase I (bottom).
SuperScript IV VILO Master Mix: Citations
The SuperScript V VILO Reverse Transcriptase has been built on the robust engineering of the previous generations of SuperScript VILO Reverse Transcriptases, which have been widely cited in several peer reviewed research publications. In five years, between 2019 and 2024, it has been cited in more than 3,000 publications.
| Research area | Use | Reference |
|---|---|---|
| Antimicrobial resistance | Synthesize cDNA for the relative measurement of RecA, a marker for resistance. | Stefan CP, Blancett CD, Huynh KA (2024). Relative quantification of the recA gene for antimicrobial susceptibility testing in response to ciprofloxacin for pathogens of concern. Sci Rep 14(1):2716. doi: 10.1038/s41598-024-52937-0 PMID: 38302590. |
| Cancer | Study expression of genes involved in a hypoxia pathway of triple negative breast cancer. | Dragonetti M, Turco C, Benedetti A et al. (2024). The lncRNAMALAT1-WTAP axis: a novel layer of EMT regulation in hypoxic triple-negative breast cancer. Cell Death Discov 10(1):276. doi: 10.1038/s41420-024-02058-4 PMID: 38862471. |
| Cancer | Synthesize cDNA for RT-qPCR to measure changes in gene expression. | Wang H, Chu F, Zhang XF et al. (2023). TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs. Cell Death Dis 14(1):64. doi: 10.1038/s41419-022-05537-7 PMID: 36707511. |
| Cancer | Synthesize cDNA from tissues and cell lines for RT-qPCR studies. | Wang Z, Yang X, Chen D et al. (2024). GAS41 modulates ferroptosis by anchoring NRF2 on chromatin. Nat Commun 15(1):2531. doi: 10.1038/s41467-024-46857-w PMID: 38514704. |
| Infectious disease and obesity | Study expression of inflammatory and immune gene expression in obese versus normal ferrets post-flu infection. | Meliopoulos V, Honce R, Livingston B et al. (2024). Diet-induced obesity affects influenza disease severity and transmission dynamics in ferrets. Sci Adv 10(19). doi: 10.1126/sciadv.adk9137 PMID: 38728395. |
| Immunology (asthma) | Quantitate N-cadherin levels. | Pereira NL, Schaible N, Desai A et al. (2024). N-cadherin antagonism is bronchoprotective in severe asthma models. Sci Adv 10(48). doi: 10.1126/sciadv.adp8872 PMID: 39612338. |
| Immunology (asthma) | Synthesize DNA from colon samples to measure gene expression of intestinal epithelial recovery and inflammation. | Dimopoulou C, Guerra PR, Mortensen MS et al. (2024). Potential of using an engineered indole lactic acid producing Escherichia coli Nissle 1917 in a murine model of colitis. Sci Rep 14(1):17542. doi: 10.1038/s41598-024-68412-9 PMID: 39080343. |
| Immunology & infectious disease | Measure ACE2 expression from low input bronchoalveolar lavage cells. | Magnen M, You R, Rao AA et al. (2024). Immediate myeloid depot for SARS-CoV-2 in the human lung. Sci Adv 10(31):eadm8836. doi: 10.1126/sciadv.adm8836 PMID: 39083602. |
| Immunology & tissue repair | Measure gene expression in sorted cells. | Jung H, Kim DH, Díaz RE et al. (2024). An ILC2-chitinase circuit restores lung homeostasis after epithelial injury. Sci Immunol 9(100). doi: 10.1126/sciimmunol.adl2986 PMID: 39423283. |
| Neuroscience | RT RNA from single cells prior to ddPCR. | Scalmani P, Paterra R, Mantegazza M et al. (2023). Involvement of GABAergic interneuron subtypes in 4-aminopyridine-induced seizure-like events in mouse entorhinal cortex in vitro. J Neurosci 43(11):1987–2001. doi: 10.1523/JNEUROSCI.1190-22.2023 PMID: 36810229. |
| Plant viral infection | Detect and measure viral infection in plant samples. | Salo W, Considine JA, Considine MJ (2024). Influence of mixed and single infection of grapevine leafroll-associated viruses and viral load on berry quality. Tree Physiol 44(5). doi: 10.1093/treephys/tpae035 PMID: 38501881. |
| Plant biology | Synthesize cDNA from plant samples. | Hata Y, Ohtsuka J, Hiwatashi Y et al. (2024). Cytokinin and ALOG proteins regulate pluripotent stem cell identity in the moss Physcomitrium patens. Sci Adv 10(35). doi: 10.1126/sciadv.adq6082 PMID: 39196946. |
| Stem cell | Measure gene expression from RNA isolated from zebrafish embryos. | Pessoa RC, Collins JM, Yang S et al. (2024). Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells. Science 385(6714). doi: 10.1126/science.adn1629 PMID: 39264994. |
| Viral research | Amplify SARS-CoV-2 whole genome for Illumina sequencing. | Botelho-Souza LF, Nogueira-Lima FS, Roca TP et al. (2021). SARS-CoV-2 genomic surveillance in Rondônia, Brazilian Western Amazon. Sci Rep 11(1):3770. doi: 10.1038/s41598-021-83203-2 PMID: 33580111. |
SuperScript IV VILO Master Mix FAQs
Find tips, troubleshooting help, and resources for common questions about the SuperScript IV VILO Master Mix.
Can’t find your question?
SuperScript V VILO Master Mix is a versatile cDNA synthesis Master Mix that can be used for gene expression analysis with RT-qPCR, spanning several research fields such as oncology, immunology, and cell therapy. It can be used in advanced RNA analysis such as RNA sequencing and transcriptomics. It synthesizes high quality cDNA from difficult samples such as FFPE, blood, inhibitor-containing, GC-rich, and low input RNA.
SuperScript V VILO Master Mix (Cat. No. 11800001) contains a blend of oligo(dT) primers and random hexamer primers. SuperScript V VILO Master Mix, Primer-Free (Cat. No. 11900001) does not contain primers.
Yes. Gene-specific primers can be used with SuperScript V Reverse Transcriptase (Cat. No. 19090050) and SuperScript V VILO Master Mix, Primer-Free (Cat. No. 11900001).
Yes. SuperScript V Reverse Transcriptase and SuperScript V VILO Master Mixes use a DTT-free formulation and do not require users to add DTT during reaction setup.
In SuperScript V VILO Master Mix products with genomic DNA removal, RNA samples are first treated with ezDNase enzyme for 2 min at 37°C. The reverse transcription master mix is then added directly to the same tube before cDNA synthesis.
Yes. An additional no-RT control can be prepared by heating SuperScript V VILO Master Mix at 95°C for 1 min to inactivate the reverse transcriptase before use.
Resources
For Research Use Only. Not for use in diagnostic procedures.