name: rtqpcr-primer-design description: Advanced RT-qPCR primer and probe design with specialized support for cross-species homology mapping (e.g., Human to Vero cells) and high-GC virus optimization. Use when needing to design high-specificity qPCR assays for: (1) Standard Human/Mouse/Monkey transcripts, (2) Predicted transcripts (XM_) lacking exon data via homology mapping, (3) High-GC viral templates (e.g., PRV), or (4) SYBR Green and TaqMan probe-based assays. Designed by ZJU PhD. acceptLicenseTerms: true

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: Uses multiple anchor points and sliding-window alignment to map exon-exon junctions from a well-annotated ortholog (e.g., Human NM_) to a predicted target transcript (e.g., Vero XM_). This ensures gDNA-safe design even when target annotations are missing.
2. High-GC Template Optimization: Implements the full SantaLucia (1998) Nearest-Neighbor thermodynamic model for precise Tm calculation, essential for templates with >70% GC content (e.g., Pseudorabies virus).
3. TaqMan & SYBR Green Support:
   • TaqMan: Automatically designs probes with Tm 8-10°C higher than primers, following standard real-time PCR design rules (no 5' G, no poly-G runs).
   • SYBR: Strict 3' quality control and secondary structure screening to minimize primer-dimers.
4. Off-target Defense: Supports an optional --offtarget transcript to check for 3' end mispriming, ensuring high specificity in mixed cDNA samples.

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 junction mapping.
3. Automated Design: Execute scripts/design_qpcr_assay.py with specific parameters:
   • --target: Target accession (e.g., NM_001256799)
   • --homolog: Human ortholog for mapping (e.g., NM_001256799 for a monkey gene)
   • --offtarget: Accession for 3' specificity check (e.g., a homologous gene in the same family)
4. Validation: Review the top-scoring sets based on Tm, GC, 3' quality, and gDNA safety.

Technical Standards

• Tm Calculation: SantaLucia (1998) Nearest-Neighbor (NN) model with salt and terminal correction.
• gDNA Defense: Primers are required to span exon-exon junctions by at least 5bp on each side for stability.
• 3' Quality: Strict GC-clamp control and mismatch prevention.

Designed by ZJU PhD @ Bio-chat Community