Can Power SYBR™ Green PCR Master Mix be substituted directly for the SYBR™ Green PCR Master Mix?

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Answer

The Power SYBR Green PCR Master Mix (P/N 4368577/ 4367659/ 4368706/ 4368702/ 4368708/ 4367660) is a new and improved formulation of the regular SYBR Green PCR Master Mix (P/N 4344463/ 4309155/ 4364344/ 4364346/ 4312704/ 4334973). This new master mix contains a more purified AmpliTaq™ enzyme and improved buffer formulation compared to regular SYBR™ Green PCR Master Mix. You may need to re-validate your assays in order to start using the Power SYBR™ Green PCR Master Mix. For more information on validating your reaction with the Power SYBR™ Green PCR Master Mix, please refer to the Power SYBR™ Green PCR Master Mix Protocol (P/N 4367218).

Answer Id: E2486

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What are the requirements for a relative quantification qPCR experiment?

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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.

Answer Id: E7496

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How can RNA standards be generated to perform absolute quantitation for RNA targets?

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It is generally not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step. Therefore, in-vitro transcribed RNA is commonly used to prepare standards for the absolute quantitation of RNA targets. This would involve the cloning of the target of interest into an in-vitro transcription plasmid, performing in-vitro transcription, then purifying the resulting cRNA so that the DNA plasmid cannot serve as a PCR template. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the RNA.

Relative quantitation of gene expression methods require less up-front preparation and provide a fold-change value instead of an absolute quantity result. For many researchers, absolute quantities are not a necessary parameter to measure, and therefore relative quantitation is a much more attractive approach to studying gene expression via real-time PCR. For more information on relative quantitation of gene expression, please refer to our Technical Reference Library in the Technical Resources section of our website.

Answer Id: E1380

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Can I do a melt curve with a TaqMan™ assay?

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

Answer Id: E7493

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How much first-strand cDNA should be used in a PCR reaction?

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The volume will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 5 μL of the first-strand reaction in a 50 μL PCR reaction (i.e., 1/10 volume). More than 10% may inhibit downstream reactions. In general, for gene expression you will want to start with 1-100 ng of cDNA per well.

Answer Id: E7483

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

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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.

Answer Id: E7506

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What should I do if I have extra peaks in my melt curve? What does this mean?

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Since SYBR™ Green can bind to any double-stranded DNA product, it is important to check your melt curves for the number of peaks. When primers are specific, you should see only one peak. Sometimes you may get extra peaks in a melt curve, which could be due to primer-dimers, a nonspecific product, or gDNA contamination. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=4QPyVcpbvNw) for more information on multiple peaks in melt curves and how to deal with them.

Answer Id: E7502

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

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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).

Answer Id: E7497

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

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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.

Answer Id: E7494

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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?

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Answer

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 two-step. 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 gives greater flexibility. This table (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/1-step-vs-2-step.html) also provides a comparison.

Answer Id: E7486

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

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Answer

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.

Answer Id: E7507

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

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

Answer Id: E7489

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I checked the efficiency of my SYBR™ Green primer set, and it is low. What is causing this?

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Poor efficiency is typically caused by PCR inhibitors, limiting reagents, or suboptimal assay design. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool for more details at https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html. You can also review this short video (https://www.youtube.com/watch?v=T8Ovs6OC4KE).

Answer Id: E7503

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How do I set the baseline for my qPCR experiment?

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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).

Answer Id: E7509

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What is the difference in sensitivity between TaqMan™ chemistry vs. SYBR™ Green reagent chemistry?

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Sensitivity can actually be equivalent when using TaqMan™ chemistry and SYBR™ Green reagent chemistry. It might seem that a TaqMan™ assay with fluorescent signal generated by a sequence-specific probe would always be more sensitive than a SYBR™ Green reagent assay, but a poorly designed TaqMan™ assay could theoretically be less specific than a well-designed SYBR™ Green reagent assay. However, the potential for detection of primer dimers and non-specific products using SYBR™ Green chemistry is more likely to result in loss of sensitivity when attempting to quantitate lower copy numbers.

For more information on Real-Time PCR chemistries, please refer to the following Application Notes, which you can find on our website through Technical Resources, or by entering the titles in the main Search field: "Real-Time PCR Vs. Traditional PCR", "Essentials of Real Time PCR", and "Selection of Reagents for Real-Time PCR".

Answer Id: E1419

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