This white paper provides guidelines for designing and optimizing qPCR experiments consistent with MIQE standards. It emphasizes minimizing reverse transcription bias, selecting exon-spanning regions, screening for SNPs, and validating primers to avoid dimers and nonspecific amplification. Assay efficiency is critical: >90% is required for ΔΔCt calculations, otherwise a standard curve method must be used. Validation involves melting curves and 5-point dilution series. Thermo Fisher highlights its PowerTrack SYBR Green Master Mix, which improves specificity, reproducibility, and contamination control. While SYBR assays require optimization, TaqMan assays offer pre-validated, high-efficiency alternatives.

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