Megaplex™ Primer Pools, Human Pools Set v3.0

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.