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  7. Applied Biosystems™ TaqMan™ Fast Virus 1-Step Multiplex Master Mix for qPCR (No ROX)

Applied Biosystems™ TaqMan™ Fast Virus 1-Step Multiplex Master Mix for qPCR (No ROX)

Product Code. 17896481 Shop All Applied Biosystems Products
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Description

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) is designed to enable dye flexibility for multiplexing up to four different RNA/DNA targets for high-throughput RT-qPCR.

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) is designed to enable dye flexibility for multiplexing up to four different RNA/DNA targets for high-throughput RT-qPCR. The 4X formulation enhances detection of both RNA and DNA viral pathogens, even in the presence of challenging PCR inhibitors. TaqMan Fast Virus master mix is used for sensitive pathogen detection in research samples as well as multiplex gene expression studies with low target input. It has a single-tube format for consistent handling and processing and is ideal for high-throughput workflows, delivering results in <30 minutes.

Benefits of TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) include:

  • Multiplexing—optimized to detect up to four RNA/DNA viral pathogen and gene expression targets
  • High sensitivity—4X master mix to amplify RNA and DNA targets at low target input levels with six logs of dynamic range
  • Tolerant to PCR inhibitors—formulated to work in the presence of common RT-qPCR inhibitors found in blood, stool, and other complex samples
  • Dye flexibility—the formulation without ROX dye enables use of other dyes in the channel previously used to measure ROX
  • Simplified application—one-tube, one-step master mix offers a single run profile with RNA and DNA and allows easy mix-and-match of targets on a plate
  • High efficiency—increased RT-qPCR speed on fast and standard instruments

Optimized for multiplexing

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) has been validated for detection of up to four targets simultaneously. Viral pathogen research often includes complex reactions with multiplexed primers and probes and internal controls. To meet this need, we developed this master mix for higher order multiplex reactions with multiple gene expression targets (see figure below). To simplify your experiments, a single TaqMan Fast Virus master mix protocol has been developed to amplify both types of nucleic acid, so you can interrogate both RNA and DNA virus targets next to each other on the same plate using the same handling steps.

High sensitivity

Many viral pathogen research samples have very low levels of nucleic acid targets. TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) is optimized for highly sensitive detection of viral targets and low-titer pathogens (see figure). Formulated at a higher concentration, this master mix allows you to set up smaller reactions and run fast-cycling instruments and protocols while obtaining at least the same sensitivity as 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.

Consistent results in the presence of inhibitors

Research samples assayed for viral pathogens commonly include materials such as blood, dirt, and tissues that can inhibit qPCR reactions. TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) includes buffer components and proprietary additives that are optimized to handle RT-qPCR inhibitors to help ensure consistent performance and provide confidence in your results. The master mix has been tested to enable robust performance in the presence of common inhibitors, including heparin, hematin, and humic acid (see figure).

Dye flexibility

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) does not contain the ROX passive reference dye. This allows higher order multiplexing because other dyes, such as JUN (or similar emission wavelength dye) can be measured in the channel previously used to measure ROX. Because of this flexibility, TaqMan Fast Virus master mix can be used with common real-time PCR system and allows selection of the desired passive reference dye to best fit your needs.

Fast results

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) speeds your time to results and streamlines the use of your real-time PCR instruments with a fast protocol. The high-concentration formulation allows for more sample containing the target nucleic acid 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.

TaqMan Fast Virus 1-Step Multiplex Master Mix (No ROX) provides robust performance, multiplexing capabilities in the presence of common RT-qPCR inhibitors, and convenient reaction setup allow you to have more confidence in your results.

TRUSTED_SUSTAINABILITY

Specifications

Concentration 4X
Content And Storage Contains 1 x 1 mL tube of 4X RT-PCR master mix sufficient for 200 x 20 μL reactions.
The master mix contains:
• Hot-start AmpliTaq Fast DNA Polymerase
• Thermostable MMLV Reverse Transcriptase
• dNTPs
• RNase Inhibitor
• Optimized buffer

Store at -15°C to -25°C.
Detection Method Primer-probe
PCR Method 1-step RT-qPCR
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 Flex, QuantStudio™ 7 Pro, StepOne™, StepOnePlus™, ViiA™ 7 System
Multiplex Capability Up to 4-plex
Passive Reference Dye None
Quantity 1 x 1 mL
Shipping Condition Dry Ice
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 x 20 μL Reactions
Sample Type RNA, DNA
Volume 1 x 1 mL
Inhibitor Tolerance (Simple) High
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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.
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.
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.

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