RT-qPCR Primer Design (Bio-chat Series)

This skill provides a professional-grade workflow for designing high-specificity RT-qPCR primers and probes, optimized for complex biological samples.

Key Capabilities

1. Homology-based Junction Mapping: Automatically maps exon-exon junctions from a well-annotated ortholog (e.g., Human NM_) to a predicted target transcript (e.g., Vero XM_) to ensure gDNA-safe design even when annotations are missing.
2. High-GC Template Optimization: Specialized algorithms for templates with >70% GC content (e.g., Pseudorabies virus), automatically adjusting primer length and Tm weight to ensure specificity.
3. SYBR & TaqMan Support: Designs optimized primer pairs for SYBR Green and triplex/probe sets for TaqMan assays with precise Tm matching.

Workflow

1. Target Identification: Provide a NCBI Accession (NM_/XM_) or a local FASTA sequence.
2. Homology Mapping (Optional): For predicted monkey transcripts, provide a Human ortholog Accession to activate "Hardcore Mode" for exon junction mapping.
3. Automated Design: Execute scripts/design_qpcr_assay.py with specific parameters:
   • --target: Target accession
   • --homolog: Human ortholog for mapping
   • --offtarget: Accession for 3' specificity check
4. Validation: Review the top-scoring primer/probe sets based on Tm, GC, 3' quality, and secondary structure.

Technical Standards

• Tm Calculation: Nearest-Neighbor (NN) model.
• gDNA Defense: Primers must span or flank exon-exon junctions.
• 3' Quality: Strict control of 3' GC-clamp and mismatch prevention.

Designed by ZJU PhD @ Bio-chat Community