Applied Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix for qPCR

Catalog No.	Quantity
44-444-32	1 x 1 mL
44-444-34	5 x 1 mL
44-444-36	1 x 10 mL

Catalog No. 44-444-32 Supplier Applied Biosystems™ Supplier No. 4444432

Description

TaqMan Fast Virus 1-Step Master Mix is designed for fast, highly sensitive real-time RT-PCR, even in the presence of challenging PCR inhibitors.

The features of the kit have been selected to enhance virus detection on commonly used sample types. A single-tube format allows for uniform handling and processing for both RNA and DNA viruses.

TaqMan Fast Virus 1-Step Master Mix is designed for fast, highly sensitive real-time RT-PCR even in the presence of challenging PCR inhibitors. The 4X formulation provides enhanced detection of both RNA and DNA viruses and is ideal for multiplex gene expression studies, even with low target input. The TaqMan Fast Virus 1-Step Master Mix provides a single-tube format for uniform handling and processing, while delivering qPCR cycling in less than 30 minutes, helpful when working in high throughput workflows.

Key product features:

• High sensitivity—4X master mix to amplify both RNA and DNA with improved sensitivity
• Consistent results—formulated to handle common RT-PCR inhibitors found in blood, stool, and other difficult samples
• Simplified application— one-tube, one-step master mix, offering a single run profile with RNA and DNA, which allows for easy mix-and-match of targets on a plate
• Target flexibility—works in singleplex, multiplex, and with exogenous or endogenous internal controls
• High efficiency—increased qRT-PCR speed on fast and on standard instruments

Two formulations

The master mix is available in two formulations:

• TaqMan Fast Virus 1-Step Master Mix containing ROX dye as a passive reference is recommended for singleplex (1 probe) and up to triplex (3 probes) reactions
• TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) (Cat. No. 5555532), which does not contain a passive reference, is recommended for use with assays utilizing the JUN (or similar omission wavelength) dye or for higher order multiplexing (>3 targets)

High sensitivity

Many common research samples with viral nucleic acids have very low levels of target. We have optimized the master mix for high-sensitivity detection of viral targets. The higher-concentration master mix allows you to set up smaller reactions, which means that you can perform fast cycling protocols and obtain at least the same sensitivity as is expected from standard-cycling qPCR. Alternatively, larger sample input amounts can be added to standard reaction volumes for more accurate quantification of low-titer samples (figure below).

Consistent results in the presence of inhibitors

Research samples commonly assayed for viruses include blood, dirt, and tissues. Buffer components and proprietary additives in the TaqMan Fast Virus 1-Step Master Mix have been optimized to handle RT-PCR inhibitors (figure below) to help ensure consistent performance even with these difficult samples, so that you can be more confident in your results.

Flexibility in targets

It is common for labs to test for both RNA and DNA viruses in a variety of samples. To simplify your experiments, a single TaqMan Fast Virus 1-Step Master Mix protocol has been developed to assay both types of nucleic acid, so you can perform RNA and DNA virus queries next to each other on the same plate using the same handling steps (figure below). Additionally, because virus research often includes multiplexed primers and probes and internal reaction controls, we have optimized the master mix to work with multiple target amplicons (figure below).

Fast results

The TaqMan Fast Virus 1-Step Master Mix speeds your time to results and maximizes the use of your real-time PCR instruments with a fast protocol. The high-concentration formulation allows for more target nucleic acid sample to be added in the smaller reaction volumes required to run fast protocols. This enables you to maintain sensitivity with low-titer research samples while improving speed and throughput (figure below).

TaqMan Fast Virus 1-Step Master Mix provides a reliable and efficient reagent for real-time RT-PCR of virus samples that does not sacrifice accuracy. Its robust performance in the presence of common RT-PCR inhibitors and its convenient and flexible reaction setup allows you to have more confidence in your results.

Order Info

Shipping Conditions: Dry Ice

Specifications

• Content And Storage: Contains 1 x 1 mL tube of 4X RT-PCR master mix sufficient for 200 reactions. Components:
  • AmpliTaq™ Fast DNA Polymerase, UP
  • Thermostable MMLV Reverse Transcriptase
  • dNTPs
  • RNase Inhibitor
  • ROX™ dye (passive reference)
  
  Store at -15°C to -25°C in the dark.
• Detection Method: Primer-probe
• Form: Liquid
• Format: Tube
• PCR Method: 1-step RT-qPCR
• Polymerase: AmpliTaq Fast DNA Polymerase
• Reaction Speed: Fast or Standard
• For Use With (Equipment): 7500 Fast System, 7500 System, 7900HT System, QuantStudio 12k Flex, QuantStudio 3, QuantStudio 5, QuantStudio 6 Flex, QuantStudio 6 Pro, QuantStudio 7 Pro, QuantStudio 7 Flex, StepOne, StepOnePlus, ViiA 7 System
• Multiplex Capability: Up to 3-plex
• Passive Reference Dye: ROX (Pre-mixed)
• Product Line: TaqMan
• Product Type: 1-Step Master Mix
• Quantity: 1 x 1 mL
• Reverse Transcriptase: M-MLV
• Shipping Condition: Dry Ice
• Sufficient For: 200 Reactions
• Target Specificity: RNA only, Both RNA and DNA
• For Use With (Application): Microbial Detection, Gene Expression, DNA Quantification, Presence/Absence
• Thermostability: Not Applicable
• Hot Start: Built-In Hot Start
• No. of Reactions: 200 Reactions
• Sample Type: Total RNA, Viral DNA, Viral RNA, mRNA
• Inhibitor Tolerance (Simple): High

Frequently Asked Questions (FAQs)

What can I do to improve the sensitivity of my qPCR assay?

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

How do I set the baseline for my qPCR experiment?

Most times your instrument software can automatically set a proper baseline for your data. Check out our short video, Understanding Baselines, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=5BjFAJHW-bE).

How do I set the threshold for my qPCR experiment?

In most cases your instrument software can automatically set a proper threshold for your data. Check out our short video, Understanding Thresholds, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=H_xsuRQIM9M).

I am not getting any amplification with my TaqMan Assay or SYBR Green primer set. What is causing this?

There could be several reasons for no amplification from an assay or primer set. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/no-amplification.html) for more details.

I am getting amplification in my no-template control (NTC) wells in my qPCR experiment. What is causing this?

There could be several reasons for amplification in a NTC well. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/amplification-no-template-control.html) for more details.

My amplification curves have a funny shape in my qPCR experiment. What is causing this?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html) for more details.

What can I do if the amplification of my target gene is later than expected for my qPCR experiment?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool for more details (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html).

Can I use my SYBR Green primers for a TaqMan assay?

It may be possible to use your SYBR Green primers for a TaqMan assay, depending on how they were designed. You would have to design a separate probe to use with your existing primers. Please refer to the guidelines in this manual (https://tools.thermofisher.com/content/sfs/manuals/cms_041902.pdf) on “Manually Designing Primers and Probes” for the next steps. If you have Primer Express Software, you can use that software to design a probe. Please note that restricting the design using the predesigned SYBR primers may not allow for a successful probe design.

Do I have to normalize my samples for comparative Ct experiments?

Comparative Ct experiments use an endogenous control gene to normalize the cDNA input. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=jst-3hD_xFQ) for more details on how this works. For a protocol workflow, please refer to our Guide to Performing Relative Quantitation of Gene Expression (https://tools.thermofisher.com/content/sfs/manuals/cms_042380.pdf).

What are the requirements for a relative quantification qPCR experiment?

In a relative quantification experiment, you will need to identify an endogenous control and a reference (or calibrator) sample. An endogenous control is a gene that does not change in expression across all the samples in your study. A reference sample is the sample that you are comparing all others to. This is often the untreated, or control, sample. Please see our Relative Gene Expression Workflow bulletin (https://tools.thermofisher.com/content/sfs/brochures/cms_075428.pdf) for more step-by-step guidelines on how to design your experiment.

What are the requirements for a standard curve qPCR experiment?

In a standard curve experiment, you must generate a standard curve for each target gene. The standards should closely represent the sample (i.e., RNA for RNA input, plasmid or gDNA for DNA input). This reference (http://www.ncbi.nlm.nih.gov/pubmed/11013345) is a good review of standard curves and the experimental setup. You can also review this short video (https://www.youtube.com/watch?v=mE5ieko9_RQ) on standard curve experiments.

What is the difference between absolute quantification (AQ) and relative quantification (RQ)? How do I choose which method to use?

Absolute quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e., copy number). Unknowns can then be compared to the standard curve and a value can be extrapolated. Absolute quantification is useful for quantitating copy number of a certain target in DNA or RNA samples. The result usually is a number followed by a unit, such as copy number and ng, etc.

Relative quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. For example, in a drug treatment study you could compare a treated to an untreated sample. The quantity of the calibrator is not known and cannot be measured absolutely. Therefore the calibrator (untreated sample) and samples (treated samples) are normalized to an endogenous control (a gene that is consistently expressed among the samples) and then compared to each other to get a fold difference. Relative quantification is useful for quantitating messenger RNA levels. Since the result is a fold change or ratio, it is not followed by a unit.

The method that you choose will depend on the type of data you need from your experiment. You can find more information here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

Can I do a melt curve with a TaqMan assay?

No. A TaqMan probe, once cleaved, cannot be re-quenched. Therefore a melt curve does not apply when using a TaqMan assay.

What is the difference between TaqMan and SYBR Green methods of detection?

TaqMan and SYBR Green chemistries are two different methods of detection for qPCR. Please see this detailed comparison of these two approaches (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/taqman-assays-vs-sybr-green-dye-for-qpcr.html). You can also watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=fkUDu042xic) on how TaqMan assays work.

How many replicates do I need to run for my qPCR experiment? What recommendations do you have for plate setup?

Please view this short video (https://www.youtube.com/watch?v=eIaPGhOjBQo), which explains some best practices for replicates and plate setup.

What are the different phases of a qPCR reaction?

Check out this short video (https://www.youtube.com/watch?feature=player_embedded&v=4sXPUbIrh3A) to understand the different phases of the PCR reaction and why they are important.

I'm trying to decide between purchasing a one-step or two-step RT-PCR kit. Can you review the advantages and disadvantages of each?

One-step RT-PCR is convenient and less prone to contamination, as there is less opportunity for pipetting error. This method is also faster than the two-step process. However, the cDNA cannot be archived, and fewer genes can be analyzed. Two-step RT-PCR gives you the ability to archive cDNA, analyze multiple genes, and offers greater flexibility. Learn more about the difference between one-step and two-step RT-PCR on this page Onestep vs Twostep RT-PCR.

Can Ct's greater than a cut-off be considered valid results?

Yes they can. However, it is important to recognize the true linearity and detection limits of your assay: Ct values above the cut-off can indicate non-specific amplification, unless your NTC is a true- no-target control, and you have run a statistically significant number of replicates. Any results with Ct above the recommended cut-off need to be validated with individual assays on plates.

What is a good Ct cut-off for the TaqMan MicroRNA Array Cards and TaqMan Advanced miRNA Array Cards? In other words, beyond what Ct should I not trust the data?

The typical Ct cut-off on TaqMan Array Cards is 32, which is equivalent to Ct 35 on a plate (10 µl reaction). Previous studies show that if you use pre-amplification, a Ct cut-off of 29 or 30 can be used to reduce numbers of false positives (see Technical Note Optimized protocols for human or rodent microRNA profiling with precious samples). To ensure that you have selected a correct cut-off, you should run replicates of the same sample and use Ct cut-off before you see an increase in the Standard Deviation.

Is there a TaqMan PCR Core Reagents kit for the Fast Real-Time PCR System?

No. The only Fast reagents that we offer are the TaqMan Fast Universal PCR Master Mix (2X) for single plex use, and Fast Advanced Master Mix for greater sensitivity and multiplexing use.

Can I use TaqMan Fast Universal PCR Master Mix for both one-step and two-step RT-PCR?

TaqMan Fast Universal PCR Master Mix is recommended for two-step RT-PCR, but for one-step reactions, we recommend using TaqMan Fast Virus 1-Step Master Mix as the preferred reagent.

How can I make a no-reverse transcriptase control with a TaqMan Fast Virus 1-Step Master Mix for qPCR?

A no-RT control can be prepared by adding template, master mix, and assay in an Eppendorf tube and then heat inactivating the RT enzyme at 95 degrees C in a water bath. Check the protocol for the time needed for this step for your particular master mix during your thermal cycling heat inactivation stage. Then you can pipette the sample on to your plate and run with your samples as usual.

Why is there unexpected amplification when I use TaqMan Fast Virus 1-Step Master Mix in my RT-PCR?

Most likely there was amplification of genomic DNA. We recommend that you run a no-RT control reaction to confirm that there was genomic DNA contamination:
- Inactivate the RT enzume by incubation the Master Mix at 95 degrees C for 5 min.
- Allow the Master Mix to cool to room temperature.
Note: The PCR hot-start mechanism will reactivate.
We also recommend treating the RNA sample with DNAse I.
For Custom TaqMan Gene Expression Assays, you can also design an assay that spans an exon-exon boundary. See Custom TaqMan Assays Design and Ordering Guide (Pub. No. 4367671).

Why is the Ct low and merges with the background signal when I use TaqMan Fast Virus 1-Step Master Mix in my RT-PCR?

When automatic baseline is used, the software raises the threshold bar to avoid the elevated baseline. We recommend that you use a manual Ct, then manually adjust the threshold.

Why is the amplification plot truncated when I use TaqMan Fast Virus 1-Step Master Mix in my RT-PCR?

Most likely the baseline was set too high. We recommend that you rest the lower value of the baseline range or use an automatic baseline.

Why does the amplification plot display S-shaped curves when I use TaqMan Fast Virus 1-Step Master Mix in my RT-PCR?

Most likely the RT-PCR Reaction Mix was not thoroughly combined. We recommend that you repeat the assay, and follow instruction for mixing the reagents. Please see the assay user guide for mixing instructions.

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