TaqMan™ TaqMan™ Gene Expression Master Mix

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.

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).

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.

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).

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.

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.

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.

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

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.

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

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.

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.

TaqMan Gene Expression Master Mix has been tested with TaqMan PreAmp Master Mix (Cat. Nos. 4391128, 4488593) and High Capacity cDNA Reverse Transcription Kit (Cat. Nos. 4368813, 4368814).

Not necessarily. Performance comparisons should not focus on a direct comparison of Ct. Instead, the basis for comparison should be objective performance criteria, such as linear dynamic range and average PCR efficiencies calculated from the slope of a serial dilution of target.

Here are the important features of the TaqMan Gene Expression Master Mix:

• High sensitivity
• Improved co-amplification of two targets (duplexing capability)
• Good specificity
• Robust, consistent amplification of difficult target sequences (GC and AT-rich targets)
• High quality

TaqMan Gene Expression Master Mix contains UDG (uracil-DNA-glycosylase). UDG refers to a superfamily of enzymes comprising six sub-families. Family I UDG enzymes are called UNG, after the uracil-N-glycosylase gene. The terms UDG and UNG are commonly used interchangeably because they perform the same function in qPCR, namely to remove Uracil from dU-containing DNA to prevent carryover contamination from previous PCRs.

Yes, this is possible. However, note that all Applied Biosystems real-time PCR master mixes (including the TaqMan Gene Expression Master Mix) contain ROX dye as the passive reference dye. The software used with the Bio-Rad real-time PCR instruments does not calculate/normalize the signal between the reporter dye and ROX dye. Thus, when the TaqMan Gene Expression Master Mix is used on a Bio-Rad real-time PCR instrument, ROX dye will no longer work as a passive reference dye. Additionally, if you want to use Texas Red dye as a reporter dye on a Bio-Rad real-time PCR instrument, we do not recommend using an Applied Biosystems real-time PCR master mix because the emission wavelength of Texas Red dye is the same as that of ROX dye.

For SYBR Green applications, we have other validated master mixes, e.g., PowerUp SYBR Green Master Mix (Cat. No. A25741).