Invitrogen™ SuperScript™ IV VILO™ Master Mix

Product Code.	Includes	No. of Reactions	unitSize
15574395	Kit only	50 Reactions	50 reactions
15584395	Kit only	500 Reactions	500 reactions
15523145	Kit with ezDnase	50 Reactions	50 reactions
15595209	Kit with ezDnase	500 Reactions	500 reactions

Product Code. 15574395 Supplier Invitrogen™ Supplier No. 11756050

Description

SuperScript IV VILO (SSIV VILO) Master Mix, available with or without ezDNase, is a reaction master mix designed for fast, sensitive, and reproducible cDNA synthesis in RT-qPCR applications.

SuperScript IV VILO (SSIV VILO) Master Mix, available with or without ezDNase, is a reaction master mix designed for fast, sensitive, and reproducible cDNA synthesis in RT-qPCR applications. The inclusion of ezDNase enzyme further accelerates the RT-qPCR workflow through an extremely simplified genomic DNA removal step.

SuperScript IV VILO Master Mix features include:
• Fast RT reactions in just 10 minutes
• Convenient one-tube cDNA reaction master mix for two-step RT-qPCR
• High yields reduce Ct values by more than two cycles ahead of all other reverse transcription reagents
• Efficient reaction even with low template amounts and suboptimal purity samples
• gDNA removal in as few as two minutes (SSIV VILO Master Mix with ezDNase only)

SSIV VILO Master Mix elevates the trusted VILO technology to the next level with the highly processive and thermostable SuperScript IV Reverse Transcriptase and further optimized buffer. These components enable efficient cDNA synthesis at higher temperatures and in less time. SSIV VILO master mix provides superior cDNA yield and sensitivity even with suboptimal purity or scarce templates. It is your new tool for more efficient and reproducible RT-qPCR.

SuperScript IV VILO Master Mix with ezDNase is available for better removal of genomic DNA and 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 results in more cDNA in less time with less pipetting, less variation, less reaction inhibition, less gDNA interference, and with less sample than other cDNA synthesis kits or master mixes for RT-qPCR. Additional ezDNase may also be purchased separately.

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

Specifications

• Concentration: 5X
• Content And Storage:

  • SuperScript IV VILO Master Mix (200 μL)
  • SuperScript IV VILO Master Mix 'No RT' Control (200 μL)
  • Nuclease-free water (1.25 mL)
  
  Store at -15°C to -25°C.
  
  

• Detection Method: qPCR
• Format: Master Mix
• GC-Rich PCR Performance: High
• Reaction Speed: 10 min.
• Technique: Reverse Transcription
• Optimal Reaction Temperature: 50°C
• Quantity: Each
• Reverse Transcriptase: SuperScript IV
• Ribonuclease H Activity: Reduced
• Shipping Condition: Dry Ice
• For Use With (Application): Real Time PCR (qPCR)
• Final Product Type: First-Strand cDNA
• No. of Reactions: 50 Reactions
• Reaction Format: Master Mix
• Reagent Type: Reverse Transcription
• Size (Final Product): Up to 10 kb
• Starting Material: RNA
• Includes: Kit only

Frequently Asked Questions (FAQs)

Which Reverse Transcription kit can I use for FFPE samples?

For RT-qPCR with FFPE samples, we recommend SuperScript IV VILO Master Mix. This mix offers enhanced performance with degraded RNA and inhibitor containing samples.

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

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 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

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 RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.

Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.

Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).

Do you offer a one-step RT-qPCR kit containing SuperScript IV RT?

SuperScript IV RT is not recommended for one-step RT-qPCR applications. However, it is included in end-point one-step RT-PCR kits: https://thermofisher.com/ssiv-onestep

For one-step RT-qPCR, we recommend SuperScript III Flash Reverse Transcriptase or TaqPath RT-qPCR Master Mix products.

What control do you recommend using with the Oncomine TCR Beta-LR Assay?

We recommend using T Cell Leukemia (Jurkat) Total RNA (Cat. No. AM7858) as a control. T cell Leukemia (Jurkat) Total RNA is derived from a cell line consisting of a single T cell clonotype. Running the Oncomine TCR Beta-LR Assay on Jurkat Total RNA should enable detection of a single clonotype.

Are there any BED files for the Oncomine TCR Beta-LR Assay?

Unlike other AmpliSeq panels, there are no BED files and no reference is needed. All downstream analysis takes place in Ion Reporter Software.

Can I quantitate the Oncomine TCR Beta-LR Assay library with the Ion Library Equalizer Kit?

We have tested the Ion Library Equalizer Kit with this kit for library quantitation and it does function, but manually balancing library input into the Ion Chef instrument is preferred.

What version of Torrent Suite Software and Ion Reporter Software can be used with the Oncomine TCR Beta-LR Assay?

Torrent Suite Software 5.6 and Ion Reporter Software 5.6 are needed to generate reports. In Torrent Suite Software 5.6, two Planned Run templates are provided for use with the Oncomine TCR Beta-LR Assay.

Can I run the Oncomine TCR Beta-LR Assay on the Ion PGM System?

The Oncomine TCR Beta-LR Assay has been developed and verified for use on the Ion GeneStudio S5 Series System. The assay can be used with the Ion PGM System, however, this instrument configuration is not supported.

What are the components of the Oncomine TCR Beta-LR Assay?

Here are the components of the Oncomine TCR Beta-LR Assay:

- Oncomine TCR Beta‑LR Panel
- Ion AmpliSeq Library Kit Plus
- Ion Select Barcode Adapters

What is the concentration of the primer pool in the Oncomine TCR Beta-LR Assay?

The primer pool is at 5X concentration.

What are the amplicon read lengths in the Oncomine TCR Beta-LR Assay?

The Oncomine TCR Beta-LR Assay enables long-read RNA sequencing (up to 400 bp amplicon length) for complete characterization of the T cell receptor Beta (TCR Beta) chain, including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. This enables allele-specific interrogation of TCR Beta to potentially predict drug-mediated adverse events.

How many primer pools does the Oncomine TCR Beta-LR Assay kit contain?

The primers are provided as a single pool.

With the Oncomine TCR Beta-LR Assay, how many samples can I multiplex on each chip?

Up to 16 samples can be multiplexed on an Ion 530 chip, up to 4 samples can be multiplexed on an Ion 520 chip, and up to 4 samples can be multiplexed on an Ion 318 chip.

What research sample types are compatible with the Oncomine TCR Beta-LR Assay?

A variety of research sample types including fresh-frozen tissue, whole blood, and sorted T cells are compatible with the Oncomine TCR Beta-LR Assay.

Why are immunologists and immuno-oncologists interested in T cell repertoire sequencing research?

T cell repertoire sequencing helps:

- To characterize diversity and study features of T lymphocytes in blood and infiltrated tumors
- To study variable gene polymorphisms implicated in autoimmune disease or immune-mediated adverse events
- To study the production and function of therapeutic or engineered T cells

How many samples can I analyze with the Oncomine TCR Beta-LR Assay?

This kit is good for 24 samples.

What does the Oncomine TCR Beta-LR Assay measure?

The Oncomine TCR Beta-LR Assay is an RNA-based next-generation sequencing (NGS) research assay that enables the characterization of the T cell receptor Beta (TCR Beta) sequences including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. The assay measures T cell repertoire diversity as well as clonal expansion, and allows for identification of allele-specific polymorphisms.

What is the difference between SuperScript IV RT and the RT enzyme used in SuperScript IV VILO Master Mix?

The SuperScript IV VILO Master Mix contains SuperScript IV RT, a ribonuclease inhibitor, and a helper protein. The helper protein helps to increase the efficiency of the reverse transcription reaction and thus improve cDNA yield. This master mix formulation allows for a simpler reaction setup with less pipetting and less variation between samples.

How many reactions of SuperScript IV VILO 'No RT' Control are included with the SuperScript IV VILO Master Mix?

50 reaction packs (Cat. Nos. 11756050 and 11766050) include 200 μL of 'No RT' Control sufficient for 50 reactions. 500 reaction packs (Cat. Nos. 11756500 and 11766500) include 1 mL of 'No RT' Control sufficient for 250 reactions.

Why did you use oligo (dT)18 plus random hexamer primers for SuperScript IV VILO Master Mix whereas the SuperScript VILO Master Mix contains only random hexamer primers?

This is to help ensure that the bias from using either oligo (dT)18 or random hexamer primers alone will be eliminated. Although the data indicates that using random hexamers alone enables quantitation just as good as the combination of the two primers, this may improve data quality.

How does SuperScript IV VILO Master Mix perform with GC-rich RNA templates?

SuperScript IV VILO Master Mix often permits better cDNA synthesis results compared to other cDNA synthesis kits and master mixes. It has increased thermostability, allowing the RT incubation temperature to be raised up to 65 degrees C to deal with the GC-rich or structurally complex RNA templates.

What is the recommended reaction temperature for SuperScript IV VILO Master Mix?

The recommended reaction temperature for SuperScript IV VILO Master Mix is 50 degrees C. For GC-rich or structurally complex RNA templates, RT incubation temperature may be increased up to 65 degrees C.

What are the key performance improvements of SuperScript IV VILO Master Mix over the SuperScript VILO Master Mix and SuperScript VILO kits?

SuperScript IV VILO Master Mix uses SuperScript IV Reverse Transcriptase (RT) in the optimized master mix formulation, whereas SuperScript VILO Master Mix and SuperScript VILO kits use SuperScript III Reverse Transcriptase. SuperScript IV RT has a significantly improved performance profile over SuperScript III RT in thermostability, sensitivity, yield, processivity, and the ability to synthesize cDNA efficiently from a wide variety of RNA samples, even those of suboptimal purity and integrity.

Which applications can SuperScript IV VILO Master Mix be used for?

SuperScript IV VILO Master Mix is an upgrade from SuperScript VILO Master Mix and cDNA Synthesis Kit. It is designed to be used for cDNA synthesis in the two-step RT-qPCR applications for gene expression analysis.

For Research Use Only. Not for use in diagnostic procedures.