Superior cDNA synthesis performance in RT-qPCR applications

Invitrogen SuperScript IV VILO Master Mix is a first-strand cDNA synthesis reaction mix for two-step RT-qPCR. The master mix format elevates the trusted VILO technology (Variable Input, Linear Output) to the next level by combining further optimized buffer conditions with with highly processive and thermostable Invitrogen SuperScript IV Reverse Transcriptase (RT). The master mix offers exceptional performance features while maintaining superior linearity across the broadest range of input RNA.

Highlights

  • Super-efficient—Ct values earlier by on average 2 cycles compared to other RT reagents, in a 10-minute reaction
  • Super-strong—Reliable results even with degraded or inhibitor-containing RNA samples
  • Super-reliable—Improved RT-qPCR data reproducibility due to single-tube master mix format
  • Super-safe—Integrated, easy, and RNA-friendly genomic DNA (gDNA) removal

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SuperScript IV VILO Master Mix advantages

3 levels of control enable high RT-qPCR data reproducibility and accuracy

1

The SuperScript IV VILO Master Mix includes all the reaction components in pre-mixed formulation. You only need to add the RNA template and water to complete the reaction mix. With this all-in-one format, pipetting steps are minimized, which in turn helps to improve RT-qPCR data reproducibility and avoid contamination.

2

The SuperScript IV VILO Master Mix No-RT control (all master mix components without the RT enzyme) allows you to monitor the extent of potential gDNA contamination and to avoid misleading results.

3

ezDNase removes contaminating gDNA while preserving high RNA quality and RT-qPCR data accuracy.

Reliable performance across an extended range of RNA input

SuperScript IV VILO Master Mix includes proprietary helper protein which improves the interaction between SuperScript IV RT and the RNA template for superior sensitivity and extended linearity across the broadest range of RNA input (Figure 1).

Excellent linearity in the RT step of RT-qPCR means you will:

  • Obtain the same relative target RNA representation in your cDNA, regardless of its abundance
  • Accurately perform data normalization, even if the amount of starting material for your target of interest and for the reference target differ
  • Get greater accuracy in your RT-qPCR data by reducing this common bias

Figure 1. Extended 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 linear 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.

Ct values earlier by on average 2 cycles compared to other RT reagents

In RT-qPCR analysis of a broad range of target transcripts using low initial RNA input, SuperScript IV VILO Master Mix provided the highest efficiency, delivering greater cDNA yields and lower Ct values compared to 8 other cDNA synthesis reagents (Figure 2).

Higher cDNA synthesis efficiency in RT-qPCR applications means you can:

  • Archive cDNA for future studies
  • Achieve better RT-qPCR sensitivity to work with low RNA input, low-copy targets or degraded RNA
  • Use less RNA input in your RT-qPCR workflow
Highest efficiency across a broad range of targets
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 gene targets indicated. Delta Ct values (∆Ct= Ct – Ct SuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and on average 2 cycles lower Ct values than other reagents tested.

Reliable performance with challenging RNAs 

Many cDNA synthesis products for qPCR work optimally only with ideal, intact RNA samples but fail when challenged with suboptimal RNAs. Even with degraded or inhibitor-containing RNA, robust SuperScript IV VILO Master Mix offers superior performance (Figures 3A and 3B).

Figure 3A. Superior performance with inhibitor-containing RNA.  cDNA synthesis was performed using 100 ng total 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 – Ct SuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and lowest Ct values in presence of all reaction inhibitors.

Figure 3B. Superior 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 – Ct SuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and lowest Ct values with degraded RNA.

Fast, easy, and RNA-friendly gDNA removal 

All RNA purification methods, including protocols with on-column DNAse digestion, fail to remove gDNA completely. 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 is able to degrade single stranded DNA such as primers and cDNA, and therefore needs to be inactivated or removed before cDNA synthesis. This is commonly done using EDTA or other processes that can damage or reduce yields of RNA (Figure 4A).

SuperScript IV VILO Master Mix with double stranded DNA-specific Invitrogen ezDNase enzyme allows efficient, fast, and easy gDNA elimination (2 minutes at 37°C). Since ezDNase is thermolabile, it is inactivated at standard SuperScript IV RT cDNA synthesis temperature (50°C), eliminating the need for a separate inactivation step and enabling the highest 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 affected (lowered) RNA integrity and/or yields.

Figure 4B. Complete gDNA decontamination with ezDNase. 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 including treatment with ezDNase for 2 minutes at 37°C. ezDNase effectively removed gDNA and resulted in no target amplification.

Dramatically simplified workflow

Due to high processivity of SuperScript IV RT in the SuperScript IV VILO Master Mix, cDNA synthesis reactions are significantly faster (10 minutes) in comparison to reactions performed with traditional RT enzymes (60 minutes). In addition, the protocol for gDNA removal with ezDNase takes only 2 minutes and does not require a separate enzyme inactivation step. The workflow for cDNA synthesis and gDNA removal with the SuperScript IV VILO Master Mix is therefore significantly shorter than with traditional systems (Figure 5).

Figure 5. A timeline comparison between the SuperScript IV VILO Master Mix cDNA synthesis workflow including ezDNase treatment (top) and the traditional cDNA synthesis workflow with DNase I (bottom).

 

Resources

Frequently Asked Questions

Find tips, troubleshooting help, and resources for common questions about SuperScript IV RT and SuperScript IV VILO Master Mix

Reverse transcription education

Review educational resources for reverse transcription basics, cDNA synthesis, enzyme selection, troubleshooting tips, and cDNA applications.