The robustness, sensitivity, and specificity of a qPCR Assay also depend on the amplicon length. This is especially true for Taqman/probe assays, whereas a SYBRGreen assay reacts robustly towards different amplicon lengths as shown in fig 1a/b.

It is generally advised not to have different amplicon lengths within one assay.

Assay typeOptimal amplicon length
TaqMan / Hydrolysis probes80 – 100 bp
Probesup to 100 bp
SYBRGreen / EvaGreen120 – 200 bp

Fig 1a: Different amplicon lengths within one assay should be avoided

Mikeska, T., & Dobrovic, A. (2009). Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays. BMC Research Notes, 2, 112. https://doi.org/10.1186/1756-0500-2-112

Fig 1b: Taqman and SYBRGreen assays require different amplicon sizes

Mikeska, T., & Dobrovic, A. (2009). Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays. BMC Research Notes, 2, 112. https://doi.org/10.1186/1756-0500-2-112