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View additional product information for TaqMan™ Gene Expression Assay (FAM) - FAQs (4453320, 4351368, 4448892, 4351370, 4331182, 4351372)
77 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 order it as a custom assay. Since the assay is discontinued, it can not be ordered on-line. You will have to call or email the order to genomics@appliedbiosystems.com, and include the assay ID. The assay will be synthesized once the order is received. It will be designed and priced as a Custom TaqMan Gene Expression Assay so you will need to choose the scale.
It means that you are probably detecting genomic DNA (gDNA) in your sample. It could be the method you are using to prepare your RNA. Our website has information and products to help you eliminate genomic DNA contamination. Also, if you are using an assay ID with the suffix _s, it will, by definition, detect gDNA. Assays ending with the suffix _s are designed within a single exon. If there is gDNA present in the sample, you must treat with DNase.
TaqMan Gene Expression Assays with “_s1” as assay ID suffix are the assays whose primers and probes are designed within a single exon, and therefore, will detect genomic DNA. However, the application of these assays in gene copy number studies has not been validated. Other gene expression assays will not detect gDNA, let alone the region of variance.
The 18S rRNA is highly conserved among eukaryotes. We have several configurations of assays for this gene: P/N 4331182, Assay ID: Hs99999901_s1; PN 4319413E, 4310893E, 4333760T, 4333760F, 4308329. You can use any of these for both human and mouse samples, as well as samples from any other eukaryotic species.
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".
The PDARs for several genes were simply renamed as the TaqMan Endogenous Controls and are available in the different tube sizes in FAM/MGB and VIC/MGB dye and quencher combinations. Those genes are: 18S rRNA; GAPDH; ACTB; TBP; PGK1; GUSB; PPIA; B2M; RPLP0; and TFRC.
The TaqMan Gene Expression Assays that were created as replacements for the PDARs were designed with exactly the same sequence as the PDAR. Additionally, the assays are formulated with the same primer and probe concentration in a 20X solution and are available in 250 reaction size. There is virtually no difference between the PDAR and its replacement assay.
Yes, we have maintained a TaqMan Gene Expression Assay ID for each PDAR that was available in the past. This assay uses the exact same sequence as the PDAR, and is denoted by a series of five “9's” in the assay ID (example: Hs99999070_m1 is the old PDAR for the gene VEGF).
The TaqMan Gene Expression Assays are the improved alternative for the PDARs. With over 400,000 pre-designed assays for human, mouse, and rat designed over most exon-exon junctions of transcripts in the public RefSeq database, chances are good that you will find the right assay for your experiment. Many assays are available for each gene because assays are designed for each transcript. Therefore, TaqMan Gene Expression Assays will enable you to detect different expression levels for splice variants of genes.
The catalog number of a TaqMan Gene Expression assay indicates its reporter, its size, and its availability. Assay ID, in turn, indicates the sequences of the primers and probe of a specific assay. This means that two different assays, with different IDs, can have the same catalog number, and that a single assay can have different catalog numbers.
Find additional tips, troubleshooting help, and resources within our TaqMan Protein Assay Support Center.
The TaqMan assays are shipped at ambient temperature and are stable at ambient temperature for a few weeks. As long as they are stored away from light, they are still safe to use. This white paper, TaqMan Assays Shipped at Ambient Temperature Reduce Environmental Impact and Retain Their Quality and Stability, discusses the testing we have performed.
Find additional tips, troubleshooting help, and resources within our Gene Expression Analysis & Genotyping Support Center.
The sequences for probes and primers of our predesigned TaqMan assays are proprietary. If you need the sequence to publish the results obtained with TaqMan assays, you can find more information about publishing results using the following links below:
Primer Sequence Disclosure: A Clarification of the MIQE Guidelines
Publish Real-Time PCR Results with Confidence
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
They are delivered in TE, pH 8.0 (Cat. No AM9849 or J75793.AE).
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
The TaqMan® Gene Expression Assay (FAM) (Cat. Nos. 4351368, 4351370, 4351372, 4331182, 4448892, 4453320) maps to the following:
5' - EWSR1 exon 7 (on transcript NM005243.4)
3' - FLl1 exon 8 (on transcript NM002017.5)
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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
Once a TaqMan Gene Expression Product has been found, for example Hs00174318_m1, click on "view details" button, then look at the number of RefSeqs under Interrogated Sequences. The list of transcript IDs listed are all of the transcripts that the assay Hs00174318_m1 will detect. If the splice variant that you are interested in is listed, then this assay will work for your study.
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.
TaqMan® Gene Expression Assays are shipped at ambient temperature, but we recommend storing them at -15 degrees C to -25 degrees C upon arrival.
Currently, only Inventoried TaqMan Gene Expression, microRNA, and Advanced miRNA Assays can be ordered in the TaqMan Low Density Array. If you need other assays in the array format, you can consider our Custom Plating options.
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.
We recommend performing four replicates of each sample.
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.
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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.
This is very unlikely, as an NTC test is performed for every round of assay manufacture. Any assay failing this test is thrown out and re-synthesized and tested. Only assays passing all of our QC tests are put into inventory and are available for sale.
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.
There are two reasons an assay won't be found on the website: 1) an assay does not yet exist for the transcript of interest, or 2) the search criteria were insufficient.
It is easiest to search for an assay using a RefSeq ID, hCG, or hCT. If searching by gene symbols, it is best to omit hyphens, dashes, etc. Also, it is recommended to limit the number of characters to 3 and append an asterisk (*), which is the wildcard search symbol. If an assay is not found using RefSeq IDs, it should be assumed that the assay is currently in the design/manufacturing pipeline and will be available on the web in the near future. High homology within certain gene families can slow the design process because it is harder to make gene-specific assays. If you do not find your particular gene expression assay, you can order a Custom TaqMan Gene Expression Assay. For more information, review the Custom TaqMan Assays -Design and Order Guide (https://tools.thermofisher.com/content/sfs/manuals/cms_042307.pdf).
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The context sequence is 25 nucleotides that includes the probe sequence. The context sequence is available only after the purchase of an assay and is provided in the AIF (Assay Information File). You can find this file online at www.thermofisher.com/taqmanfiles using your Sales Order Number and the Rack ID located on the packaging.
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.
The TaqMan Gene Expression human endogenous control Assays are designated by an assay ID that starts with Hs999999xx_yy. The primer/probe sequences for these assays are the same as the FAM/MGB and VIC/MGB TaqMan Endogenous Controls, but are manufactured in our high-throughput oligo factory. Unlike regular TaqMan Gene Expression Assays, the probes of the endogenous controls may not span an exon junction - however, the amplicon will span an exon junction. There are some genes with both TaqMan Endogenous Control primer/probe designs and TaqMan Gene Expression primer/probe designs. Example: Gene Symbol TFRC, Refseq: NM_003234, Target assay: Hs00174609_m1, Endogenous control: Hs99999911_m1. The endogenous controls will not detect gDNA (except 18S) and are tested for this criterion with each round of manufacturing. The 18S rRNA assay (Hs99999901_s1) will detect gDNA.
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.
Assay suffixes are assigned at the time of assay design. We do not recommend choosing an assay solely based on the suffix; always be sure to check the full assay details. An "_m1" assay is one in which the probe spans an exon-exon junction and should not detect genomic DNA, but we recommend inspecting the assay details to verify, as some "_m1" assays could possibly detect gDNA due to updates in the reference genome sequence. An "_s1" assay is one in which the primers and probe lie within a single exon. This assay will detect genomic DNA. In a "_g1" assay, the assay probe spans an exon junction, or the assay primers and probe lie within a single exon. “_g1” assays will or might detect gDNA. This could be due to on-target gDNA detection, or off-target gDNA detection. For any assay that has the potential to detect gDNA, we recommend treating your RNA samples with DNase I and running no-RT controls in your qPCR to explicitly test for gDNA contamination.
Please see below for descriptions of all gene expression assay suffixes:
_m: An assay whose probe spans an exon junction.
_s: An assay whose primers and probes are designed within a single exon. Such assays, by definition, detect genomic DNA.
_g: An assay that will or might detect genomic DNA. The assay primers and probe may also be within a single exon.
_mH, _sH, _gH: An assay that is designed to a transcript belonging to a gene family that has high sequence homology. These assays are designed to yield a 10- to 15-CT difference between the target gene and the gene with the closest sequence homology. This means that the assay detects the target transcript with 1000- to 30,000-fold greater discrimination (sensitivity) than the closest homologous transcript, if both transcripts are at the same copy number in a sample.
_u: An assay whose amplicon spans an exon junction, and whose probe binds completely in one of the spanned exons.
_ft: An assay designed to detect fusion transcripts that result from chromosomal translocation. One primer and the probe are on one side of the fusion transcript breakpoint, and the second primer is on the other side of the fusion transcript breakpoint. This assay does not detect gDNA.
_at: An assay that is designed to detect a synthetic RNA transcript with a unique sequence that lacks homology to current annotated biological sequences
Note: An assay ID beginning with “Hs999999…” and ending in “_m1” identifies a TaqMan Gene Expression Assay that amplifies a region spanning an exon junction, although the associated probe does not span the junction. For example, the exon boundary information for assay Hs99999903_m1 is 1-1, indicating that the probe targets a region within exon 1, not the exon junction itself. Although the probe binds within a single exon, the amplicon spans exons 1-2 (the forward primer and probe are in exon 1, but the reverse primer is in exon 2).
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
Yes. For duplexing, we have VIC-labeled probes with primer limited concentrations (VIC PL) available from our pre-designed assay selection. The primer limited assay is used to detect the most abundant gene. For guidance on multiplexing, see the TaqMan Multiplex PCR Optimization User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/taqman_optimization_man.pdf).
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.
Linear range has to be determined empirically. A dilution series of several logs needs to be tested and evaluated to determine this range. Evaluation of the linear range can be determined visually or by mathematical methods. For more information, refer to Validation of Compendial Methods. This document describes the elements under The Current Good Manufacturing Practice Regulations (21 CFR 211.194(a)) requiring that test methods meet proper standards of accuracy and reliability. Some of the measures are obtained by assessing the following parameters for your PCR assays: Accuracy, Precision, Specificity, Limit of Detection, Limit of Quantitation, Linearity and Range, Ruggedness and Robustness. Copies of these guidelines can be obtained through the FDA. This document describes the criteria to determine and qualify the Linear Dynamic Range of your assay.
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.
Fluorescent probes should be stored protected from light to prevent photobleaching. They should be wrapped in aluminum foil or kept in a cardboard box in a -20°C freezer.
To minimize freeze/thaw cycles, we recommend you aliquot the stock probe dilution into small “working” volumes needed for one reaction and store at -20°C. Prior to use, the solution should be lightly vortexed and spun down using a bench-top centrifuge.
For more information on how to dilute your custom primers and probes, refer to the following guide: "Reconstituting and Diluting Primers and TaqMan Probes" (Cat. No. 4370992). You can find a copy on our website by searching the title or part number.
The concentration of the TaqMan probe is provided at a concentration of 100 µM (pmol/µL), if pre-diluted. This information can also be found on the data sheet that accompanies a probe order. If the probe is sent dried, the total amount present in the vial will be listed on the data sheet in picomoles, relative to the scale that was ordered.
The extinction coefficient of VIC is 30,100 cm-1M-1.
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.