Real-Time qPCR: Design, Optimisation & Data Analysis

Overview

Quantitative PCR (qPCR) measures DNA/RNA abundance in real time using fluorescent reporters such as SYBR Green or TaqMan probes.

Primer Design Rules

• Amplicon size: for efficient amplification.
• GC content: .
• Melting temperature (Tm):
• Avoid runs of >4 identical nucleotides and self-complementarity.

Reference Gene Selection

Choose two or more stable reference genes validated in your experimental system. Common choices include GAPDH, ?-actin, 18S rRNA, and HPRT1. Use geNorm or NormFinder to assess stability.

The ??Ct Method

?Ct(sample)    = Ct(gene of interest) ? Ct(reference gene)
??Ct           = ?Ct(treated) ? ?Ct(control)
Fold change    = 2^(???Ct)

Assumption: The PCR efficiency of the target and reference amplicons must be equal (~100 %).

Troubleshooting Common Issues

• High Ct values
• Non-specific amplification
• Poor reproducibility