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查看更多产品信息 TaqMan™ siRNA Assay - FAQs (4440880, 4440879, 4440878, 4440877)
22 个常见问题解答
The expiration date for your TaqMan® siRNA Assay (Cat. No. 4440877) can be found on the Certificate of Analysis (COA).
You can find the COA for a specific lot number by using the Documents and Certificates search page or by filling out a Certificate of Analysis (COA) or Certificate of Origin (COO) Request Form.
Please be advised that according to TaqMan® Assays qPCR Guarantee Terms & Conditions:"Guarantee claims should be made within 12 months of delivery or by the specified expiry date noted on packaging or associated product documentation, whichever is shorter. Any such claims not made by the customer within the applicable warranty period will be forfeited. TaqMan® Assay qPCR Guarantee is limited to the buyer of the product from Thermo Fisher and is not transferable."
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
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. Try excluding the well, then 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 the 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 possible solutions:
- 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).
Amplification in the no-RT control can be due to the cDNA template or amplicon being contaminated. Please follow established PCR good laboratory practices.
Please review the following possible causes and solutions:
- Contamination occurred. We recommend that you run 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.
- 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:
- The reagents were not mixed properly. Increase the length of time that you mix the reagents. We recommend that you confirm your mixing process by running a replicate 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.
- The threshold was not set correctly. Set the threshold above the noise level and where the replicates are tightest. 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.
- Ct is not the most appropriate analysis for the data. We recommend that you try using relative threshold (Crt) analysis. See Introduction to Gene Expression Getting Started Guide (Pub. No. 4454239).
Please review the following possible causes and solutions:
- The endogenous control is not consistently expressed across the samples. Ensure that the endogenous control is consistently expressed in your sample type. We recommend that you use an alternative endogenous control such as a nonvariable miRNA.
- 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.
It is possible that the reagents are contaminated with amplicons. 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.
This can be caused by incorrect dyes selected for each target. Check the dyes selected for each target, then reanalyze the data.
This is most likely due to the sample evaporating. We recommend that you check the seal of the adhesive film for leaks before running the plate on the real-time PCR instrument.
Please review the following possible causes and solutions:
- Precipitation present in the buffers. Before preparing reactions, please make sure that you mix the Master Mix thoroughly to produce a homogenous solution.
- Reagents are 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 sample does not have enough copies of the target RNA. To confirm the results, we recommend that you rerun the sample using the same assay and/or rerun the assay using more of the sample. Also 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. We recommend checking your pipetting equipment and/or technique.
- Incorrect dyes were selected for each target. In this case, check the dyes selected for each target, then reanalyze the data.
Please review the following possible causes and solutions:
- The baseline was set improperly. This can potentially be corrected by switching from automatic baseline to a manual baseline (or vice versa), and/or increasing the upper or lower value of the baseline range. Please see your real‑time PCR system user guide for procedures on setting the baseline.
- The sample quality was poor. In this case, you can perform a quality check on the sample and then re-extract the sample if needed.
- There were different concentrations caused my imprecise pipetting. To remedy this, please follow accurate pipetting practices.
- The reagents or equipment are contaminated. Please ensure that your workspace and equipment are cleaned properly.
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 andextension 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 sample are not diluted.
- The baseline and/or threshold was improperly set. This issue can potentially be resolved by switching from an automatic baseline to a manual baseline (or vice versa), and/or lowering the threshold value to fall within the appropriate range.
Please see your real-time PCR system user guide for procedures on setting the baseline and threshold.
- 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 TaqMan Small RNA Assays only) The design or synthesis of the custom assay failed. Ensure that the sequence you submitted is correct, and check for an alternative trascript or splice variant.
Most likely there is little or no Master Mix present in the reaction due to inaccurate pipetting. Please follow accurate pipetting practices when setting up reactions.
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.
Here are some possible causes and solutions:
- The baseline was set too high and some samples have Ct values lower than the baseline stop value. In this case, we recommend switching 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 will increase the Ct value.
Amplification in the negative control well indicates that the reagents or the cDNA template are contaminated. Please follow established PCR good laboratory practices to prevent contamination.
Poor reproducibility across technical replicates indicates that the reagents were not adequately mixed. Please ensure that all samples and reagents are mixed well.
The Ct value for the NTC can be <35 for the following reasons:
- There are non-specific interactions between primers. For NTC information on a specific assay, see the Megaplex Assay Performance File.
- The cDNA template is contaminated. Follow established PCR good laboratory practices to prevent contamination.