Reverse transcription combined with quantitative PCR, or RT-qPCR, is one of the most common RNA analysis methods. It allows detection and quantification of RNA for gene expression analysis, pathogen detection, disease research, and other applications. In the RT-qPCR workflow, two common approaches can be used: one-step or two-step RT-qPCR.
In a two-step RT-qPCR workflow, RNA quantification is performed in two separate reactions. First, sample RNA is converted to complementary DNA, or cDNA, in the reverse transcription step; the first cDNA strand generated is then amplified and quantified by qPCR. One of the key advantages of the two-step process is flexibility with reaction setup and the possibility to analyze multiple targets from an RNA sample.
Since the accuracy of gene expression quantification largely depends on cDNA quality and quantity, the reverse transcription step is critical for successful RT-qPCR. Invitrogen SuperScript IV VILO Master Mix is a first-strand cDNA synthesis reaction mix designed specifically for two-step RT-qPCR. The master mix contains the proven Invitrogen SuperScript IV Reverse Transcriptase, leverages the VILO (Variable Input, Linear Output) technology, and has optimized buffer conditions for exceptional performance and linearity across the broadest range of input RNA.
Additionally, the SuperScript IV VILO Master Mix provides 3 levels of control that enables data accuracy and reproducibility in RT-qPCR reactions:
SuperScript IV VILO Master Mix is known for its reliability, efficiency, robustness, ease of gDNA removal, and workflow simplicity. Click on the attributes below to see supporting data.
Linearity across ten orders of magnitude for input RNA
Four times more cDNA, Ct values lower by two cycles
High cDNA yields with degraded or inhibitor-containing RNA samples
Fast, easy, and RNA-friendly gDNA removal
Time-saving, simplified workflow
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.
The cDNA, generated from low RNA input, showed that the SuperScript IV VILO Master Mix had the highest efficiency, delivered greater cDNA yields, and lower Ct values when compared to eight other commercially available RNA to cDNA kits (Figure 2). With SuperScript IV VILO Master Mix, the average yield of cDNA was four times greater, and the Ct values were on average by two cycles earlier. The benefits of higher cDNA synthesis efficiency include:
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.
Often 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 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.
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).
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Find tips, troubleshooting help, and resources for common questions about the SuperScript IV VILO Master Mix. Can’t find your question? Search the FAQ database
For RT-qPCR applications, we recommend using the Invitrogen SuperScript IV VILO Master Mix. The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps, and therefore reduces variation in the data. SuperScript IV Reverse Transcriptase, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitor products.
The SuperScript IV VILO Master Mix contains SuperScript IV RT, a ribonuclease inhibitor, and a helper protein. The helper protein aids in increasing the efficiency of the reverse transcription reaction and thus improves cDNA production. This master mix formulation enables a simpler reaction setup with less pipetting and less variation between samples.
SuperScript IV VILO Master Mix uses SuperScript IV RT in the optimized master mix formulation, whereas SuperScript VILO products are based on SuperScript III RT. Compared to SuperScript III RT, SuperScript IV RT has significantly improved performance due to higher thermostability, processivity, and the ability to synthesize cDNA efficiently from a wide variety of RNA samples, even those of suboptimal purity and integrity.
The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50°C). The additional inactivation step is therefore not required in RT-qPCR applications.
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