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View additional product information for TaqMan™ Non-coding RNA Assay - FAQs (4426963, 4426961, 4426962)
32 product FAQs found
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.
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.
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.
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.
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.
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.
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.