Assay Design

• Primer3 and Primer-BLAST workflows; specificity screening against the target genome
• Secondary structure: hairpins, self-dimers and cross-dimers in multiplex panels
• Hydrolysis probe versus intercalating dye chemistry; reporter and quencher selection
• Balancing multiplex reactions and designing internal amplification controls

Quantification

• Amplification efficiency, the 90–110% acceptance window, and what falls outside it
• Cq determination, baseline and threshold setting, and their effect on results
• ΔΔCq relative quantification and reference-gene validation with geNorm or NormFinder
• Establishing limit of detection and limit of quantification defensibly

Digital PCR

• Partitioning and Poisson statistics: absolute counts without a standard curve
• Choosing dPCR over qPCR for low-abundance targets and inhibited matrices
• Rare-variant detection, copy-number assessment and residual disease monitoring

Validation

• Analytical sensitivity and specificity; in-silico inclusivity and exclusivity
• Cross-reactivity panels and interference testing against the intended matrix
• Control strategy: no-template, positive, extraction and internal controls
• Contamination control: dUTP/UNG carryover prevention and workflow zoning
• MIQE-compliant documentation and the verification versus validation distinction

Practice

• Diagnosing no amplification, late Cq, and non-sigmoidal curves from the trace
• Detecting inhibition with spike-in controls and dilution series
• Melt-curve interpretation and distinguishing true product from primer-dimer
• Plate-level QC, run acceptance criteria and reporting for an accredited lab