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  7. Applied Biosystems™ AgPath-ID™ One-Step RT-PCR Reagents

Applied Biosystems™ AgPath-ID™ One-Step RT-PCR Reagents

Catalog No. AM1005
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Description

AgPath-ID™ One-Step RT-PCR Reagents are designed for sensitive, robust amplification of RNA targets using a rapid single-tube TaqMan™ real-time reverse transcription PCR (RT-PCR) strategy.

AgPath-ID™ One-Step RT-PCR Reagents are designed for sensitive, robust amplification of RNA targets using a rapid single-tube TaqMan™ real-time reverse transcription PCR (RT-PCR) strategy.

  • Recommended for RNA pathogen amplification
  • Consistent amplification of RNA targets with high specificity and sensitivity
  • Optimized to work with your target-specific primers and probes
  • Contains ROX for quantitative fluorescent signal normalization

AgPath-ID™ One-Step RT-PCR Reagents are configured for fast and simple reaction setup. The reactions are assembled in a single tube, minimizing sample handling errors and expediting set-up time. Once assembled, results are available in approximately one hour. The 25X RT-PCR Enzyme Mix included in the kit contains highly efficient ArrayScript™ Reverse Transcriptase, a mutant MMLV RT that produces high cDNA yields, and AmpliTaq Gold™ polymerase, the preferred hot-start DNA polymerase for specific target amplification. The 2X RT-PCR Buffer has been optimized for efficient, robust reverse transcription and PCR includes the passive reference dye, ROX™ Dye, for quantitative fluorescent signal normalization.

Specifications

Concentration 25X
Content And Storage Sufficient for 100 reactions of 25 μL, contains:
• 1375μL 2X RT-PCR Buffer
• 110μL 25X RT-PCR Enzyme Mix
• 1.75mL Nuclease-free Water.
Description Works on all AB platforms, but is optimized for the 7500.
Detection Method Primer-probe
Format Tube
GC-Rich PCR Performance High
PCR Method 1-step RT-qPCR
Polymerase AmpliTaq Gold DNA Polymerase
Reaction Speed Standard
For Use With (Equipment) 7500 System, 7500 Fast System, 7900HT System, StepOne™, StepOnePlus™, ViiA™ 7 System, QuantStudio™ 3, QuantStudio™ 5, QuantStudio™ 6 Flex, QuantStudio™ 7 Flex, QuantStudio 6 Pro, QuantStudio 7 Pro, QuantStudio™ 12k Flex, QuantStudio™ Absolute Q Digital PCR System
Passive Reference Dye ROX (Pre-mixed)
Product Line AgPath-ID, Ambion
Product Type One-Step RT-PCR Reagent
Quantity 100 reactions
Reverse Transcriptase ArrayScript™
Shipping Condition Dry Ice
Sufficient For 100 Reactions
Fidelity (vs. Taq) 25 X
No. of Reactions 100 Reactions
Sample Type RNA
Volume 1.75 mL
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Frequently Asked Questions (FAQs)

With the AgPath-ID One-Step RT-PCR Reagents, I am getting signal detection in the no-template control (NTC) reaction. Why is this?

This is likely due to PCR contamination. Here are some recommendations:

- Repeat the qRT-PCR reaction with fresh reagents and decontaminated pipettors.
- Set up and run the qRT-PCR reaction in an area that is isolated from areas used for nucleic acid isolation and PCR product analysis.
- The Reverse Transcriptase enzyme contained in this kit is produced using an E. coli expression vector containing a proprietary version of the MMLV pol gene (GenBank accession no. J02255) expressed from pET-24(+). It is possible that a minimal amount of the expression vector could be carried over into the final mastermix formulation. If you are targeting MMLV, a related virus, or any of the plasmid sequence, we recommend designing primer sequences for target sequences not contained in the expression vector.
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.
Is the Detection Enhancer, for real-time PCR still included with the AgPath ID One-Step RT-PCR Reagents?

No, in an effort to improve the impact on environmental health and comply with chemical safety labeling, the Detection Enhancer component was removed from the AgPath ID One-Step RT-PCR Reagents. It is now available for purchase separately. Here are the part numbers for the stand-alone Detection Enhancer, for real-time PCR:
Cat. No. A44810: Detection Enhancer, 100 reactions
Cat. No. A44941: Detection Enhancer, 500 reactions
Cat. No. A44811: Detection Enhancer, 1000 reactions

For Research Use Only.