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View additional product information for TaqMan™ Gene Expression Assay, VIC primer-limited - FAQs (4448486, 4448485, 4448484)
36 product FAQs found
Yes, it is possible. The amplicons do not have to be the same length in order to duplex the assays. You need to use different reporter dyes on the probes in multiplex - they should have a substantial difference in maximum emission wavelength to be effective. A classic example is to use FAM and VIC for the 2 probes. Depending on the expression of the targets, you may also need to limit the primer concentration for one of the assays. This is common when duplexing a target and endogenous control (the control probe is VIC labeled and the primer concentration is decreased so the target assay can properly compete for use of enzyme and dNTPs).
The final primer concentration for the primer-limited Endogenous Controls is 150 nM for the forward primer and 150 nM the reverse primer. Usually, the endogenous control genes are abundantly expressed. The limited primer concentration will force the PCR of the endogenous control to plateau early, so that it leaves enough dNTPs and enzyme for lower expressed target genes to be amplified.
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.
The following FAM MGB labeled assay can be used (Cat. No. A50137; Assay ID: Pa04930436_g1).
Find additional tips, troubleshooting help, and resources within our Gene Expression Analysis & Genotyping Support Center
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.
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.
You can view TaqMan Gene Expression Assay product coverage on the Assay details page. To do this, search for your gene of interest, for example "Cox". On your results page, select the "Assay ID" for a particular TaqMan Gene Expression Assay; for example: Hs00187909_m1. The resulting page is the assay details page and will list regions covered by the TaqMan Gene Expression Assay.
The primers that are included with assays having SKU numbers 4453320, 4448892, 4331182, 4351372, 4351368, 4351370, 4448489, 4448490, 4448491, 4331348, 4332078, 4332079, 4448508, 4448509, 4448510, 4441114, 4441117, 4441118, 4448514, 4448515, 4448516, 4426961, 442696, and 4426963 are at non-limiting concentrations. For multiplexing purposes, see the primer limiting assays with SKU numbers 4448484, 4448485, 4448486, 4448487, 4448488, 4448492, 4448511, 4448512 and 4448513 .
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The TaqMan Gene Expression Assays accelerate human disease research studies by providing quantitative gene expression assays for all human genes. They are an essential tool for screening more array hits faster and easier. The assays utilize the 5' nuclease chemistry, which provides maximum sensitivity with a wide dynamic range. This allows for standardization of gene expression data resulting in direct data comparison between labs.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
No, genomic DNA is not an intended template of these assays, but some of them may detect it along with cDNA depending on their design. Check the Assay Design section which will indicate whether the probe spans an exon junction (will not detect gDNA) or if the primers and probes are designed within a single exon (will detect gDNA).
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The NCBI Reference Sequence project (RefSeq) provides reference sequence standards for the naturally occurring molecules. The NCBI RefSeq project maintains the current knowledge of sequence information and the corresponding biology, and avoids the redundancy often present in the GenBank database archive.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
Several hundred TaqMan Gene Expression Assays have been tested for PCR efficiency. When tested across a 6-log range, all assays approached 100% efficiency (+/- 10%). This statistically significant number of tested assays validates the design pipeline. For more information on the efficiencies of TaqMan Gene Expression Assays please consult the Application Note entitled "Amplification Efficiency of TaqMan Assays-on-Demand Gene Expression Products"
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
We do not functionally test all TaqMan Gene Expression Assays. However, a statistically significant number of human, mouse, and rat assays have been functionally tested. The same assay development pipeline was used for all Gene Expression assays customized for the specific genome. The informatics pipeline includes extensive in silico QC, ensuring specificity and reproducibility.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
Content associated with each assay is gathered from a variety of public and private sources. These sources are continuously being updated. In an effort to keep our data current, we perform in silico QC of the assay and its associated content approximately every 6 months. This automated process may prohibit an individual assay from being displayed on the web at a given time.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
It is possible that there is more than one assay for a particular gene, but they are designed across different locations on the gene. Information on where the assay is located and which RefSeqs/GenBank entries are detected can be found on the Assay Details Page. It is also possible that there are two assays for the same gene that are designed on the same location. When there are multiple options, we recommend to choose the "Best Coverage" assay (based on its selection criteria).
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.
For TaqMan Gene Expression Assays, the probe spans the splice site (exon-exon junction) for multi-exon genes. For endogenous controls labeled Hs999999xx_m1, the amplicon will span an exon junction.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
We do not recommended to use an assay across species (except 18S, HS99999901_s1). The TaqMan Gene Expression Assays were designed within a species and are not evaluated for cross-species detection. A very small number of assays may detect other species. In order to evaluate this potential, use the assay location to generate an amplicon and BLAST against the sequence of interest. Note: It is important to understand that the assay will not produce reliable results for that alternate species unless there is 100% homology to the alternate species transcript sequence.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The underlying feature of the assay design pipeline is to ensure that the assay will detect only transcripts for a specific gene. Products are gene-specific, but not necessarily transcript-specific. That is, an assay may detect more alternatively spliced transcripts from the same gene. The accession numbers for transcripts that will be detected by an assay are listed on the assay details page.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The TaqMan Assays are available for purchase on the Thermo Fisher Scientific website. A particular gene expression assay can be searched for by going to our assay search page. The available assays can be searched by accession number (NCBI RefSeq ID), gene name, gene family and functional groups and categories. For assistance on searching the site, please contact Technical Support at techsupport@thermofisher.com. Once you have found the assay(s) of interest you may add them to your shopping basket and proceed with the order.
No, the predesigned TaqMan Gene Expression Assays are only available with either a 6’ FAM- or VIC-labeled TaqMan MGB probe. The TaqMan MGB probe utilizes a non-fluorescent quencher for improved sensitivity and quantitative precision.
The TaqMan Gene Expression Assays enable researchers to conduct real-time quantitative PCR gene expression studies quickly and easily by eliminating the time-consuming processes involved in assay development, which typically include the following stages:
- Sequence selection
- BLAST analysis against transcript and genomic databases
- Primer and probe design
- Oligonucleotide ordering/manufacture
- Optimization of primer and probes concentrations
- Quality control and functional testing
The TaqMan Gene Expression Assays are a comprehensive collection of pre-designed gene expression assays designed for real-time quantitative PCR. Each assay consists of a 20X formulation of unlabeled PCR primers and a TaqMan MGB probe typically labeled with either FAM or VIC dye, and is provided in a single-tube format. Each assay has been designed for real-time detection and quantification of specific genetic sequences in RNA samples which have been converted to cDNA.
TaqMan Gene Expression Assays include:
- Two unlabeled primers: 1X final concentration is 900 nM per primer; 20X stock concentration is 18 µM per primer
- One labeled TaqMan MGB probe: 1X final concentration is 250 nM; 20X stock concentration is 5 µM
For assays available as "Primer Limited": 1X final concentration is 150 nM per primer; 20X stock concentration is 3 µM per primer
Our TaqMan Assays and Arrays search tool has a filter called Cross-Reactivity Check that enables selecting the species that you do not want your assay to detect. Assays that are excluded from the search when a species is selected may exhibit species cross-reactivity. Assays that remain will not exhibit species cross-reactivity and thus are suitable to use in a transgenic study.
We do offer two VIC labeled beta actin probes as part of TaqMan Endogenous Controls:
1) Human beta actin (VIC/ TAMRA Probe, Primer Limited) Cat. No. 4310881E
2) Human beta actin (VIC/ MGB Probe, Primer Limited) Cat. No. 4326315E
Please consult our Real-Time PCR Applications page for more information.