Blog
qPCR analysis insights, method guides, and software updates from the AnnealIQ team.
Setting Threshold and Baseline in qPCR: How Misplacement Distorts Your Fold Changes
Step-by-step guide to setting qPCR threshold and baseline correctly. Includes a worked example showing how misplacement inflates fold-change calculations, plus an auto-vs-manual decision table.
Reading qPCR Melt Curves: What Multiple Peaks Actually Mean
Systematic decision tree for interpreting qPCR melt curves with multiple peaks. Covers primer-dimers, off-target amplification, AT-rich double peaks, and when to trust vs. discard your data.
7 qPCR Primer Design Mistakes That Wreck Your Efficiency Numbers
Avoid the 7 most common qPCR primer design mistakes for Tm, GC content, amplicon size, and self-complementarity. Includes specific thresholds, fixes, and a quick-reference checklist to hit 90-110% efficiency.
The Delta Delta Ct Method Explained: Assumptions, Calculation, and When It Fails
Step-by-step delta delta Ct calculation for qPCR gene expression with a worked example. Covers the two critical assumptions (equal efficiency, ~100% efficiency), when to use the Pfaffl method instead, statistical testing on delta Ct values, and MIQE reporting requirements.
Choosing Reference Genes for RT-qPCR: Stability Testing and Common Pitfalls
How to select and validate reference genes for qPCR normalization using GeNorm, NormFinder, and BestKeeper. Covers candidate gene panels, stability thresholds, geometric mean normalization, and MIQE reporting requirements.
Calculating qPCR Efficiency: Standard Curve Method and What Poor Efficiency Means
Learn how to calculate qPCR primer efficiency from a standard curve using the slope formula E = 10^(-1/slope) - 1, with a worked example, troubleshooting guide for results outside 90-110%, and MIQE reporting requirements.