Search
Search
View additional product information for TaqMan™ Gene Expression Assay, VIC - FAQs (4448490, 4448489, 4448491)
61 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).
Yes, you can check the end product on an agarose gel. If you use the TaqMan Universal PCR Master Mix (including AmpErase UNG, Cat. No. 4304437), we recommend running the gel immediately after the PCR, since the trace amount of AmpErase UNG may digest the end product that contains double-stranded DNA with dUTP incorporated. The average length of amplicon ranges from 50-150 bp. The exact amplicon length is indicated on the detailed page of each assay on our website. You can get to that page by clicking on the Assay ID; the amplicon information is listed in the section "Assay Details".
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.
When preparing the TaqMan or Custom TaqMan Gene Expression Assays, the recommended and supported volume is 20-50 µL for a 96-well standard plate set-up; 10-20 µL for a 96-well fast plate set-up and 5-10 µL for a 384-well set-up. For more information, please consult the TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays protocols.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
For optimal performance of the TaqMan and Custom TaqMan Gene Expression Assays, use 1 to 100 ng of cDNA per 50 µL total reaction volume. For more information, please consult the TaqMan Gene Expression Assay protocol and Custom TaqMan Gene Expression Assay protocol.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
After shipment, the assay mixes should be stored at -15 C to -20 C to maximize stability. Freeze/thaws should be kept to a minimum (a maximum of 10 freeze-thaw cycles is recommended). Keep all TaqMan and Custom TaqMan Gene Expression and SNP Genotyping Assays protected from direct exposure to light. Excessive exposure to light may affect the fluorescent probes. If desired, the Assay Mix may be diluted with TE buffer or DNase-free Water. It is recommended to make working stocks from the stock solutions provided in these kits.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
No, SYBR Green dye I is not compatible with the TaqMan Gene Expression Assays or Custom TaqMan Gene Expression Assays. TaqMan Gene Expression Assays or Custom TaqMan Gene Expression Assays contain TaqMan MGB probes combined with primers at non-limiting concentrations. The probes already have detection dyes conjugated to them, and SYBR Green is not needed. For more information, please consult the protocol of TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
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.
TaqMan Gene Expression Assays are pre-designed, quantitative, human gene expression assays based on the TaqMan probe-based chemistry. The assays are formulated into a 20X mix.
Custom TaqMan Gene Expression Assays or Custom Plus TaqMan RNA Assays are designed based on the target sequences submitted by the customer, then synthesized, formulated into 20X concentration, and delivered. The target sequence information can be submitted by the customer through the online Custom Assay Design Tool. The integrity of the sequence information must be verified before the design process. When you submit a sequence, you have an option to let us run the bioinformatic evaluation of the target sequence to verify the integrity (Custom Plus) or you can run the bioinformatic evaluation yourself. This integrity verification can be accomplished through BLAST analysis or other methods of verifying sequence integrity. See the Custom TaqMan Assays Design and Ordering Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_042307.pdf) for more information.
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
Each TaqMan Gene Expression Assay consists of two unlabeled PCR primers and a FAM or VIC dye-labeled TaqMan MGB probe (with non-fluorescent quencher). All the components are combined into a 20X formulation. Purchase of a TaqMan Gene Expression Assay does not provide TaqMan PCR Master Mix.
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.
Please review the following possible causes and solutions:
-The sample evaporated. Check the seal of the adhesive film for leaks.
- The well is empty because of inaccurate pipetting. Check the calibration of the pipettes, and pipette at least 5 µL of sample.
- The well is assigned a sample or target in the plate document or experiment, but the well is empty. Ensure that the plate document or experiment is set up correctly. Then try excluding the well and reanalyzing the data.
A small ΔRn can mean that the PCR efficiency was poor. Ensure that the reagents were used at the correct concentration. Alternatively, the quantity of the cDNA may be low (a low copy number of the target). In this case, we recommend that you increase the quantity of the cDNA in the reaction.
Most likely fluorescence did not stabilize to the buffer conditions of the reaction mix.
Note: This condition does not affect PCR or the final results.
We recommend that you try the following:
- Reset the lower value of the baseline range.
- Use an automatic baseline.
- Use the relative threshold algorithm (Crt). See Introduction to Gene Expression Getting Started Guide (Pub. No. 4454239).
Please review the following possible causes and solutions:
- The reagents were not mixed properly. We suggest increasing the length of time that you mix the reagents and confirming your mixing process by running a replicate assay.
- Pipetting was inaccurate. We recommend that you check the pipette calibration, and pipette at least 5 µL of sample to prepare the reaction mix.
- The threshold was not set correctly. Set the threshold above the noise level and where the replicates are tightest. Please see your real‑time PCR system user documentation for more information about setting the threshold.
- There was a low concentration of the target of interest. Rerun the assay with more cDNA template.
Please review the following possible causes and solutions:
- The endogenous control is not consistently expressed across the samples. Please ensure that the endogenous control is consistently expressed in your sample type.
- The sample concentrations vary. We recommend that you quantify and normalize the PCR samples before running the assay.
- Pipetting was inaccurate. We recommend that you check the pipette calibration, and pipet at least 5 µL of sample to prepare the reaction mix.
Most likely the reagents are contaminated with gDNA, amplicon or plasmid clones. We recommend that you clean your workspace and equipment, then rerun the assay using new reagents. We also recommend that you run no-RT controls to rule out genomic DNA contamination, and treat the sample with DNase.
Most likely the ROX dye was not set as the passive reference. Set ROX dye as the passive reference, then reanalyze the data.
Most likely incorrect dyes were selected for each target. We suggest checking the dyes selected for each target, then reanalyzing the data.
The sample evaporated. We recommend that you check the seal of the adhesive film for leaks before running the plate on the real-time PCR instrument.
It is possible that there was precipitation in the buffers. Before preapring reactions, we recommend that you mix the Master Mix thoroughly to produce a homogenous solution.
It as also possible that the reagents have degraded. Ensure that the kits and reagents have been stored according to the instructions on the packaging and that they have not expired.
Please review the following possible causes and solutions:
- The gene is not expressed in the sample. First, confirm that the gene is expressed in the sample type or tissue type at ncbi.nlm.nih.gov/unigene. If the gene is expressed, confirm the results by rerunning the sample using the same assay and/or rerunning the experiment using more of the sample. Avoid preparing PCR reaction mixes with more than 20% reverse transcription reaction. You can also run the experiment using an alternative assay, if available, that detects a different transcript or more than one transcript from the same gene.
Note: If the recommended actions do not resolve the problem, the result may be correct.
- The sample does not have enough copies of the target RNA. To confirm the results, we suggest rerunning the sample using the same assay and/or rerunning the assay using more of the sample. Avoid PCR reaction mix with more than 20% from the reverse transcription reaction.
Note: If the recommended actions do not resolve the problem, the result may be correct.
- One or more of the reaction components was not added. Please check your pipetting equipment and/or technique.
- Incorrect dyes were selected for each target. We recommend checking the dyes selected for each target, then reanalyze the data.
Please review the following possible causes and solutions:
- The baseline was set improperly. See your real‑time PCR system user guide for procedures on setting the baseline. We recommend switching from an automatic baseline to a manual baseline (or vice versa) and/or increasing the upper or lower value of the baseline range.
- The sample quality was poor. We recommend performing a quality check on the sample, then re-extracting the sample if needed.
- There were different concentrations caused my imprecise pipetting. Please follow accurate pipetting practices.
- The reagents or equipment are contaminated. Please ensure that your workspace and equipment are cleaned properly.
Most likely little or no MasterMix is present in the reaction due to inaccurate pipetting. Please follow accurate pipetting practices when setting up reactions.
Please review the following possible causes and solutions:
- The baseline was set too high and some samples have Ct values lower than the baseline stop value. We recommend that you switch from manual to automatic baselining, or move the baseline stop value to a lower Ct. The baseline stop value should be set to a Ct 2 cycles before the amplification curve crosses the threshold. Please see your real‑time PCR system user guide for procedures on setting the baseline.
-No baseline can be set because the amplification signal is detected too early in the PCR cycles. Diluting the sample can increase the Ct value.
Please review the following possible causes and solutions:
-Genomic DNA (gDNA) contamination occured. We recommend that you improve sample extraction methods and use DNase to ensure minimal gDNA contamination of the RNA.
For custom assays, we recommend that you design an assay that spans an exon-exon boundary. Please see the Custom TaqMan Assays Design and Ordering Guide (Pub. No. 4367671).
- The cDNA template or amplicon is contaminated. In this case, please follow established PCR good laboratory practices.
Please review the following possible causes and solutions:
- Contamination occurred. We recommend that you run a no-RT control to confirm that there was genomic DNA (gDNA) contamination. We also recommend the use DNase to ensure minimal gDNA contamination of the RNA.
For custom assays, we recommend that you design an assay that spans an exon-exon boundary. Please see the Custom TaqMan Assays Design and Ordering Guide (Pub. No. 4367671).
- Too much cDNA template was added to the reaction. We recommend that you quantitate the RNA before the reverse transcription (RT) reaction. After the RT reaction, adjust the concentration of cDNA before adding it to the reaction.
- The cDNA template or the amplicon is contaminated. Follow established PCR good laboratory practices.
Please review the following possible causes and solutions:
- One or more of the reaction components was not added. Ensure that the cDNA, the assay and the Master Mix were added to the reaction plate. The passive reference fails if the Master Mix is missing.
- Incorrect dyes were selected for each target. Check the dyes selected for each target, then reanalyze the data.
- The annealing temperature was too high for the primers and/or probe. Ensure that the correct annealing and extension temperatures are set, and that the real-time PCR instrument is calibrated and maintained regularly.
- Inappropriate reaction conditions were used. Ensure that the properties and the thermal protocol are correct, then troubleshoot the real-time PCR optimization.
- The template is degraded. Determine the quality of the template, then rerun the assay with fresh template if needed. We recommend that you use use RNase-free reagents and an RNase inhibitor.
- Inhibitors are present in the reaction. To check for inhibitors, we recommend that you run a serial dilution of your sample with an expressing assay (for example, an endogenous control). If an inhibitor is present, the high concentration samples yield higher-than-expected Ct values because the samples are not diluted.
- The baseline and/or threshold was improperly set. See your real-time PCR system user guide for procedures on setting the baseline and threshold. Some possible solutions to this issue are switching from an automatic baseline to a manual baseline (or vice versa), and/or lowering the threshold value to fall within the appropriate range.
- The reverse transcription failed. We recommend checking the RNA integrity and concentration, checking for RNase activity, following the recommended thermal profile, and/or repeating the reverse transcription using new reagents.
- (Custom TaqManGene Expression Assays only) The design or synthesis of the custom assay failed. Ensure that the sequence you submitted is correct. We also recomend that you check for an alternative trascript or splice variant.
- (Custom TaqManGene Expression Assays only) The assay is designed in a variable regions of the gene transccript. Ensure that the location that is targeted by the assay is not within the 5' untranslated region (UTR), which can be highly variable
between transcripts. If the assay is designed within the 5' UTR, we recommend that you select a different assay that is within the coding region of the transcript. Otherwise, select an assay for an alternative transcriptor splice variant.
There may be interaction between the primer and probe. We recommend that you adjust the threshold manually, or select another assay from the same gene, if available.
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.
Yes, we offer two options for obtaining endogenous control assays for gene expression studies:
The first option is our specially designed TaqMan Endogenous Controls products, which are a collection of probe and primer sets that are optimized, pre-formulated, and ready-to-use specifically as control assays to save on time. Endogenous Control formulations are available as primer limited and contain probes labeled with FAM or VIC reporter dyes. This allows multiplexing of TaqMan Endogenous Controls with TaqMan target gene primer and probe sets, provided that the control gene is more abundantly expressed than the target gene.
Another option is to simply order a general TaqMan Gene Expression Assay for genes that are commonly used as endogenous controls, such as 18S rRNA, GAPDH, beta-actin, etc. The TaqMan Gene Expression Assays are biologically informative, pre-formulated assays for rapid, reliable detection and quantification of human mRNA transcripts. They come in a wide variety of reaction sizes and formats, and may be ordered with either a FAM or VIC reporter dye label with MGB quenchers. You can also specify that a VIC-labeled probe should be Primer Limited (PL) for multiplexing.