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View additional product information for Megaplex™ Primer Pools, Human Pools Set v3.0 - FAQs (4444750)
28 product FAQs found
Yes they can. However, it is important to recognize the true linearity and detection limits of your assay: Ct values above the cut-off can indicate non-specific amplification, unless your NTC is a true- no-target control, and you have run a statistically significant number of replicates. Any results with Ct above the recommended cut-off need to be validated with individual assays on plates.
The typical Ct cut-off on TaqMan Array Cards is 32, which is equivalent to Ct 35 on a plate (10 µl reaction). Previous studies show that if you use pre-amplification, a Ct cut-off of 29 or 30 can be used to reduce numbers of false positives (see Technical Note Optimized protocols for human or rodent microRNA profiling with precious samples). To ensure that you have selected a correct cut-off, you should run replicates of the same sample and use Ct cut-off before you see an increase in the Standard Deviation.
Highly variable values for endogenous controls is most likely due to biological variation. Use an alternative endogenous control such as a non-variable miRNA.
Please review the following possible causes and solutions:
- There are non-specific interactions between primers. For NTC information on a specific assay, refer to the Megaplex Assay Performance File.
- The cDNA template is contaminated. Please follow established PCR good laboratory practices.
- The preamplification product was not diluted properly before real-time PCR. Follow the recommendations in the protocol. Please see "Dilute the preamplification reaction" on page 18.
It is possible that the threshold is set too high to detect miRNA in samples with low expression. Identify an appropriate NOAMP flag threshold if the relative threshold algorithm (Crt) is used. Alternatively, identify an appropriate threshold if Ct is used.
Most likely the dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. Use the relative threshold algorithm (Crt). Crt can correct for a variable baseline.
Alternatively, use the Relative Quantification application, available on Connect. The Relative Quantification application uses Crt if the software specific to your instrument does not have the relative threshold algorithm.
Please review the following possible causes and solutions:
- Bubbles in wells. Use proper pipetting techniques to avoid introducing air into the fill reservoirs. Consider omitting the leaking wells from analysis.
- Wells leaking due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. Consider omitting the leaking wells from analysis.
IMPORTANT! Do not move the carriage back across the card. Please see "Seal the card" on page 37.
- Not all the positions in the card holder were filled before centrifuging. Ensure that all the empty positions of the card holder are filled with blank balance cards before centrifuging.
- The cards were centrifuged using a non-verified centrifuge. Use a Sorvall centrifuge to prepare cards. Please see the Resources section at thermofisher.com/taqmanarrays for a list of compatible centrifuges.
- The dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. Analyze results with the relative threshold algorithm (Crt) instead of the baseline threshold algorithm (Ct ).
Please review the following possible causes and solutions:
- Empty wells due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer.
- Empty wells due to misalignment of the TaqMan Array Card Sealer. If the TaqMan Array Card Sealer is misaligned, contact Support.
- PCR reaction mix improperly prepared. Ensure that all reaction components were added to the PCR reaction mix
No amplification can indicate empty wells due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer.
IMPORTANT! Do not move the carriage back across the card. Please see "Seal the card" on page 37.
Most likely the card was in a diagonal position during centrifugation because not all the positions in the card holder were filled. Repeat the assay with a new card and ensure that all the positions in the centrifuge card holder are filled.
Most likely some wells were filled improperly. Continue with running the card. However, consider omitting the wells associated with that fill reservoir.
Please review the following possible causes and solutions:
- The fill port is blocked. Inspect the card for blocked fill port or a pinched channel. If the fill reservoir is defective, contact Support.
- Filling is incomplete or not consistent. Centrifuge the card again for 1 min. If the filling is still incomplete after the additional centrifuge cycle, continue with running the card. However, consider omitting the wells associated with that fill reservoir.
Most likely when loading the card with PCR reaction mix, air was introduced into the fill reservoir. Inspect the affected rows after centrifuging and sealing the card. Note wells that contain bubbles, then consider omitting these wells from analysis.
The PCR reaction mixture was not correctly pipetted into the fill reservoir. Be sure to correctly pipette the entire PCR reaction mixture (100 µL) into the fill reservoir. Add more sample specific PCR reaction mix to the fill reservoir.
Most likely the PCR reaction mixture was not correctly pipetted into the fill reservoir. Be sure to correctly pipette the entire PCR reaction mixture (100 µL) into the fill reservoir. Add more sample specific PCR reaction mix to the fill reservoir, if needed.
Most likely the card was not at room temperature before being removed from the packaging. Remove condensation on the reaction wells by lightly blowing room temperature pressurized nitrogen or using an air blower on the wells.
IMPORTANT: Ensure that all water condensation is removed. The optical side of the card must be free of water condensation
Most likely the reagents or the cDNA template are contaminated. Please follow established PCR laboratory best practices.
Most likely the reagents were not adequately mixed. Ensure that all samples and reagents are mixed well.
Highly variable values for endogenous controls is most likely due to biological variation. We recommend that you use an alternative endogenous control, such as a non-variable miRNA.
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. Please ollow established PCR good laboratory practices to prevent contamination.
- The preamplification product was not diluted properly before real-time PCR. Ensure that you dilute the preamplification product according the procedure in the user guide.
Most likely, the threshold is set too high to detect miRNA in samples with low levels of expression. We recommend adjusting the threshold (Ct) or NOAMP flag threshold (Crt) to an appropriate level.
Most likely the dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. We recommend that you use the relative threshold algorithm (Crt), which can correct for a variable baseline. If the software specific to your instrument does not have the relative threshold algorithm use the Relative Quantification app on Thermo Fisher Connect.
Please review the following possible causes and solutions:
- Bubbles in wells. Use proper pipetting techniques to avoid introducing air into the fill reservoirs. Consider omitting the leaking wells from analysis.
- Wells leaking due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. We recommend that you consider omitting the leaking wells from analysis.
IMPORTANT: Do not move the carriage back across the card.
- Not all of the positions in the card holder were filled before centrifuging. Ensure that all of the empty positions of the card holder are filled with blank balance cards before centrifuging.
- The cards were centrifuged using a non-verified centrifuge. We recommend that you use a Sorvall centrifuge to prepare cards. Please see the Resources section at thermofisher.com/taqmanarrays for a list of compatible centrifuges.
- The dried-down assays on the card were reconstituted at different rates, causing a dip in the early cycles of the baseline. We recommend that you analyze results with the relative threshold algorithm (Crt) instead of the baseline threshold algorithm (Ct).
Please review the following possible causes and solutions:
- Empty wells due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. Do not move the carriage back across the card.
- Empty wells due to misalignment of the TaqMan Array Card Sealer. If the TaqMan Array Card Sealer is misaligned, contact Support.
-PCR reaction mix improperly prepared. Ensure that all reaction components were added to the PCR reaction mix.
Usually, empty wells are due to improper card sealing. When sealing the card, use a slow and steady motion to push the carriage across the TaqMan Array Card Sealer. Do not move the carriage back across the card.
Most likely the card was misaligned in the block during the instrument run. Inspect the card for crushed or distorted feet. If there are damaged feet, contact Support.
Most likely the card was misaligned in the block during the instrument run. Inspect the card for crushed or distorted feet. If there are damaged feet, contact Support.
This gradient signal indicates that the card was in a diagonal position during centrifugation because not all of the positions in the card holder were filled. We recommend that you repeat the assay with a new card and ensure that all the positions in the centrifuge card holder are filled.