Why does the negative control well show amplification when doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards?
Most likely the reagents or the cDNA template are contaminated. Please follow established PCR laboratory best practices.
Why do I have poor reproducibility across technical replicates when doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards?
Most likely the reagents were not adequately mixed. Ensure that all samples and reagents are mixed well.
When doing microRNA analysis using Megaplex Primer Pools and TaqMan Array Cards, why are the Ct values for the endogenous controls highly variable across the sample set?
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.
Why is the Ct value for the no-template control (NTC) <35 for some TaqMan microRNA Assays?
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.
Why can I not detect any miRNA in my sample using the TaqMan MicroRNA Assay?
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.