Megaplex™ RT Primers, Rodent Pool B 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.

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.