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

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

 

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.

 

 

 

 

 

 

 

 

 

C_t 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 C_t values compared to 8 other cDNA synthesis reagents.

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. 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 – CtSuperScript 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.
 

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 – CtSuperScript IV VILO) show that SuperScript IV VILO Master Mix delivered the highest cDNA yield and lowest Ct values in presence of all reaction inhibitors.

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 – CtSuperScript 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 C_t 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.

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

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.

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.

 

SuperScript™ IV VILO™ Master Mix
SuperScript™ IV VILO™ Master Mix- with ezDNase™ Enzyme

• Super-fast RT reaction in just 10 minutes and gDNA removal in 2 minutes
• Super-high yields—lower Ct values by more than 2 cycles ahead of all other reverse transcription reagents
• Super-convenient one-tube cDNA reaction master mix for two-step RT-qPCR
• Super-efficient reaction even with low template amounts and suboptimal purity samples

For even better performance, this SuperScript IV VILO Master Mix includes ezDNase (also available as a separate purchase) for genomic DNA removal to further accelerate the RT-qPCR workflow. The extremely simplified genomic DNA removal step dramatically reduces the time of the entire reverse transcription protocol and reduces possible variation in gene expression due to RNA loss or damage during conventional DNase treatment.

SSIV VILO master mix gives you more cDNA in less time with less pipeting, less variation, less reaction inhibition, less gDNA interference, and with less sample than all the other cDNA synthesis kits or master mixes for RT-qPCR.

• Source

Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability and half-life, processivity, inhibitor resistance, and to reduce RNase H activity.

• Performance and quality testing

Assayed for endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease, as well as yield and length of cDNA product.

ezDNase™ Enzyme

Invitrogen™ ezDNase™ Enzyme is a recombinant double-strand-specific DNase for the fast removal of contaminating genomic DNA from RNA preparations. It cleaves phosphodiester bonds in double-stranded DNA to yield 2–8 bp oligonucleotides with 5’-phosphate and 3’-hydroxyl termini.

Features of ezDNase Enzyme include:

• Efficient and fast genomic DNA removal
• Highly specific—no impact on RNA, cDNA, or primers in RT reactions

ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA or single-stranded DNA present in the reaction such as cDNA and primers. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55°C). These features make ezDNase a superior choice for genomic DNA removal prior to reverse transcription reactions.












When used in conjunction with SuperScript™ IV VILO™ Master Mix or other Invitrogen reverse transcription reagents, ezDNase Enzyme helps to significantly reduce the likelihood of cDNA synthesis being compromised by genomic DNA and reduces variation in RT qPCR quantification caused by RNA loss or damage during conventional DNase treatment.

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.

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