Why should I choose a SYBR Green® assay vs. an hydrolysis probe (TaqMan®) assay for microRNA real-time PCR?
Both are valid assays if performed and analyzed properly. For the analysis of mRNA expression, hydrolysis probe assays are generally thought to be more specific due to the combination of two specific primers and one specific probe. The short length of the microRNA target however, means that the primers and probe are bound to overlap to some extent, minimizing the additional specificity offered by the hydrolysis probe. For both types of assay, it is highly recommended to determine PCR efficiency using a standard curve for accurate relative quantification by the ΔΔCt method (see question 28). SYBR Green® -based assays allow for melt-curve analysis, which indicates if possible non-specific (primer-dimer, etc.) amplification is occurring and contributing to false qPCR efficiencies. This is not possible with the TaqMan® assay, thus the influence of competing amplification is not known.
