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View additional product information for Custom Plus TaqMan™ RNA Assay, FAM - FAQs (4441114, 4441118, 4441117)
46 product FAQs found
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)
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).
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).
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.
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.
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.
No, the assay is not detecting different genes. Genes often have multiple valid accession numbers, even for the exact same sequence. Even though there are different ID numbers listed, they should all represent the same gene.
The label on the TaqMan Gene Expression Assays lists the expiration date, which under proper storage conditions is 5 yrs. The assay itself is guaranteed for 1 yr upon shipmentt.
We often design multiple assays for the same target. The difference between the assay IDs is most often that the assay is designed across a different exon-exon boundary of a particular transcript sequence. You can confirm the exon-exon boundary by clicking on the individual Assay ID and then look at the Interrogated Sequence Table.
Another difference between assays for the same gene is that they detect different mRNA species or splice variants for the same gene. Ensure that the assay you select will detect your transcripts of interest.
No, the Custom TaqMan Gene Expression Assays are only synthesized with FAM dye as the reporter dye at the 5' end and MGB-NFQ as the quencher at the 3' end. Dye selection is only possible with general custom oligo synthesis.
In order to take advantage of our superior in-house assay design algorithm, we recommend that you order a small scale of the standard Custom TaqMan Gene Expression Assay with the FAM label first and validate the assay in a single-plex reaction. You can then submit the sequences of the primers and probes for custom oligo synthesis where you will have the flexibility of choosing your reporter dye.
For the 23 species that we cover, you can use the species filter to narrow your results. This is located in the right-hand column of your assay search results. Look for “Exclude Assays that Detect”. Only species that will cross-react are listed (of the 23 species we screen against). Check the species box to remove assays that would detect in that particular species.
After the primers and probe are designed for a Custom TaqMan Gene Expression Assay, they will be sent for synthesis. Certain primer and probe sequences cannot be synthesized to pass our strict Quality Control procedure. We will try one more time to synthesize the oligos. If the second synthesis fails an email will be sent, notifying you that the order failed manufacturing. You will not be billed if the assay is not manufactured.
If your assay fails during manufacturing you will need to design a new set of primers/probe and send the oligo sequences to us for custom oligo synthesis. If you have an Applied Biosystems real-time PCR instrument, you should have a copy of Primer Express software, which can be used for general primer/probe design.
For optimal results, we recommend to run the reaction plate right after setting up the reaction. If a reaction plate cannot be run within 2 hours of completing the reaction setup, then freeze/refrigerate the reaction plate until it can be loaded and run on the fast PCR instrument platform.
No, we do not recommend this approach. Since the standard TaqMan Universal PCR Master Mix is not optimized for running with fast thermal cycling, assay results will be significantly compromised.
The TaqMan Fast Universal PCR Master Mix is provided without any UNG and you should add in AmpEraseUNG to reactions separately, with the addition of an initial 2 minute cycling step at 50º C. If you prefer to have a master mix with the UNG pre-added, please use TaqMan Fast Advanced Master Mix.
In general, assays have been found to perform comparably with these reagents, but they may not provide exactly identical results.
We recommend a storage temperature of 4C and the shelf life is 9 months from the manufacturing date.
We recommend adding 10 to 100 ng of RNA converted to cDNA as your template.
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.
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.
The hot-start DNA polymerase enzyme system in the TaqMan Fast Universal PCR Master Mix does not require an activation step, and can be activated with significantly shorter hold times for the thermal cycling stages of PCR, thereby minimizing PCR run time. For more information on using this reagent, please consult the TaqMan Fast Universal PCR Master Mix (2X) protocol.
The fast master mix has demonstrated detection down to 10 copies of the RNase P gene with genomic DNA. Of course, detection limits may vary depending on factors such as assay design and sample preparation.
The following research assays are supported using the TaqMan Fast Universal and/or Advanced Fast PCR Master Mix: TaqMan Gene Expression Assays, Custom TaqMan Gene Expression Assays, Custom primers and probes designed from Primer Express software-designed quantitative assays.
While TaqMan Gene Expression Assays can be used to perform a one-step RT-PCR reaction, these assays have not been optimized for this application. TaqMan Gene Expression Assays have been designed, tested, and optimized using Applied Biosystems universal conditions for a two-step RT-PCR reaction.
The assays have not been optimized for multiplex, so we don't recommended that you use them in a multiplex reaction. If you would like to try to multiplex, follow the guidelines stated in the Application Note "Factors Influencing Multiplex Real-Time PCR".
Since the Custom TaqMan Assay design software will design oligos to either strand of the target sequence, it is possible that the probe was designed for the complementary strand. Check both strands of sequence for matches to the primer/probe sequences, making sure to check the reverse complement sequence, as well. The assay information file does state "Forward" or "Reverse" to indicate which strand was used to design the probe.
During the TaqMan Gene Expression Assays product (including TaqMan Gene Expression Assays and Custom Plus TaqMan RNA Assays) design process, we take the following steps to avoid regions of ambiguity: 1. We BLAST the primer and probe designs against transcript databases to ensure specificity to ensure the chosen assay detects only transcript(s) from the gene of interest. 2. We BLAST primer and probe designs against genome databases in order to avoid assays that detect pseudo-genes or genomic DNA.
We recommend performing four replicates of each sample.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The assay location (found on the assay details page) designates the nucleotide location that is the center of the context sequence for the associated accession number. This number can be used to generate a potential amplicon that can be used to determine homology to other sequences, and similarity to partial clone sequences (full length clones are listed on assay details page), and other sequences of interest. Using the associated accession number and an external data source (i.e. NCBI, CDS, etc.), create an amplicon by selecting the amplicon size on both sides of the assay location.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The sequence provided for the assay design may contain mismatches with sample sequences. We recommend that you perform bioinformatics analysis for assay design.
For more information, see the Custom TaqMan Assays Design and Ordering Guide (Pub.No. 4367671).
For orders placed after March 2016, assay information files can be accessed from this page: www.thermofisher.com/taqmanfiles
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
The number of reactions will depend on your final reaction volume. The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL (20X)
Medium: 750 µL (20X)
Large: 976 µL (60X)
Assuming a 20 µL final reaction volume, the small size would provide 360 reactions, the medium size would provide 750 reactions, and the large size would provide 2,900 reactions.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL
Medium: 750 µL
Large: 976 µL
Please note that the large scale is supplied in a 60X format, while the small and medium scales are supplied in 20X format.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM, VIC, or VIC primer limited.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM or VIC dye, with a MGB-NFQ quencher.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.