Blog
Guides, tutorials, and best practices for qPCR data analysis.
How to Analyze qPCR Data: A Complete Guide
A step-by-step qPCR analysis workflow: QC raw Ct values, pick a quantification method, normalize, compute fold change, run stats — done right.
March 13, 2026 ★ FeaturedWhat Happened to qbase+ — and Why I Built VoilaPCR
I went looking for qbase+ and it was gone. So I built a browser-based qPCR analysis tool that does what qbase+ did, without the pain.
June 18, 2026Mic qPCR Data Analysis: Export Options, File Formats, and How to Actually Work With Your Data
How to export data from the Bio Molecular Systems Mic qPCR cycler, what file formats you get, and how to handle compatibility issues in downstream analysis.
June 17, 2026My Fold Change Is 1.5—Is That Biologically Significant?
A 1.5-fold change by qPCR: real or noise? How to assess biological significance when your effect size is small but your p-value is fine.
June 16, 2026Manual vs. Auto Baseline Correction in qPCR: When to Override Your Software
Auto baseline works most of the time — until it doesn't. Learn when manual baseline correction matters and how to set it correctly.
June 15, 2026When to Use Welch's t-Test Instead of Student's for qPCR Data
Student's t-test assumes equal variance. Your qPCR data probably doesn't have it. Here's when and why Welch's t-test is the better default.
June 12, 2026GraphPad Prism vs Excel for qPCR Statistical Analysis: Which One Should You Actually Use?
A practical comparison of Prism and Excel for qPCR stats — what each does well, where each fails, and when you need neither.
June 11, 2026qPCR Amplification Plateau Lower Than Expected — What Happened
Low fluorescence plateau in qPCR? Here's what actually causes it — reagent depletion, inhibitors, suboptimal probe — and how to fix it.
June 10, 2026Do I Need a Standard Curve for Every qPCR Plate?
When you actually need a standard curve on every qPCR plate, when you can skip it, and how to validate the shortcut without compromising your data.
June 9, 2026Converting Rotor-Gene Data to RDML Format for Analysis
How to export Rotor-Gene Q data and convert it to RDML format for use in analysis tools, with practical steps and common pitfalls.
June 8, 2026How Many Cycles Should I Run for Gene Expression qPCR?
40 cycles is standard for gene expression qPCR, but the reasoning matters. Here's when 40 is right, when 45 helps, and when fewer is fine.
June 5, 2026Biological Replicates Showing Different Ct Value Patterns — Why It Happens and When to Worry
Biological replicate Ct values diverging? Here's how to tell if it's real biology, sample prep issues, or a problem worth troubleshooting.
June 4, 2026My qPCR Standard Curve R² Is 0.97—Is That Good Enough?
An R² of 0.97 on your qPCR standard curve looks decent but often signals a real problem. Here's how to diagnose it and when to worry.
June 3, 2026CFX96 vs QuantStudio 3: Which qPCR Machine Belongs in Your Core Lab?
A practical comparison of the Bio-Rad CFX96 and Thermo QuantStudio 3 for shared-use core labs—throughput, software, data export, and what actually matters day-to-day.
June 2, 2026Can You Use GAPDH and ACTB Together as Reference Genes?
When pairing GAPDH and ACTB as reference genes makes sense, when it doesn't, and how to evaluate their stability in your specific experiment.
June 1, 2026Standard Curve Slope of -3.1 vs -3.3: Does It Matter?
A qPCR standard curve slope of -3.1 vs -3.3 both fall in the acceptable range, but the difference changes your calculated concentrations. Here's by how much.
May 29, 2026NTC Wells Amplifying After Cycle 35—Problem or Normal?
Late Ct values in your no-template controls don't always mean contamination. Here's how to interpret NTC amplification after cycle 35 and when to actually worry.
May 28, 2026Exporting QuantStudio Data to Excel for Delta Delta Ct Analysis
How to export QuantStudio qPCR data to Excel and set up a proper ΔΔCt calculation — file formats, common pitfalls, and a faster alternative.
May 27, 2026How to Handle qPCR Technical Replicates with One Outlier
One of your three technical replicates looks off. Here's when to drop it, when to keep it, and how to make that call defensibly.
May 26, 2026Your qPCR Efficiency Is 90-110% But Results Still Look Wrong — Here's Why
Passing efficiency doesn't guarantee accurate qPCR results. Here's what else goes wrong and how to systematically find the real problem.
May 25, 2026Why Is My Reference Gene Ct Value Above 30 — And When to Worry
High Ct values for GAPDH, ACTB, or other reference genes usually point to a sample problem, not a primer problem. Here's how to diagnose and fix it.
May 22, 2026How to Set the Ct Threshold in qPCR (And Why It Matters)
Where you place the Ct threshold changes your data. Here's how to set it correctly — manually and automatically — and when the default is wrong.
May 21, 2026Primer Dimer vs Nonspecific Amplification: How to Tell the Difference
Melt curve has extra peaks? Here's how to distinguish primer dimers from nonspecific amplification — and what to do about each.
May 20, 2026One-Step vs Two-Step RT-qPCR: Choosing the Right Workflow
When should you use one-step vs two-step RT-qPCR? A practical comparison of sensitivity, throughput, flexibility, and when each workflow actually matters.
May 19, 2026Replicate Variability in qPCR: How Much Is Too Much?
How much Ct spread between qPCR replicates is acceptable? Practical thresholds, what causes variability, and when to drop an outlier.
May 18, 2026How to Calculate and Interpret qPCR Amplification Efficiency
A practical guide to calculating qPCR amplification efficiency from standard curves, what the numbers actually mean, and when efficiency matters most.
May 15, 2026What Is a Good Ct Value in qPCR? Ranges, Context, and When to Worry
Learn what Ct values actually mean in qPCR, what ranges to expect for common targets, and when a high or low Ct signals a real problem.
May 14, 2026CFX96 vs QuantStudio: Comparing qPCR Export Formats and What They Mean for Your Analysis
A practical comparison of export file formats from Bio-Rad CFX96 and Applied Biosystems QuantStudio, and how to handle each for downstream analysis.
May 13, 2026How to Validate Reference Genes with geNorm and NormFinder
Step-by-step guide to validating qPCR reference genes using geNorm and NormFinder — with real examples, interpretation tips, and common mistakes.
May 12, 2026How to Statistically Analyze qPCR Fold Change Data (Without Getting It Wrong)
Run statistics on ΔCt values, not fold changes. Here's exactly how to analyze qPCR data with t-tests, ANOVA, and proper error propagation.
May 11, 2026RT-qPCR vs qPCR: They're Not the Same Assay
RT-qPCR measures RNA via a reverse transcription step; qPCR amplifies DNA directly. Here's what actually differs and why the distinction matters.
May 8, 2026How to Export and Analyze QuantStudio qPCR Data
A practical guide to exporting qPCR data from QuantStudio software and analyzing it properly — file formats, Ct extraction, and common pitfalls.
May 7, 2026NTC Amplification in qPCR: What It Means and When to Worry
Late Ct in your no-template controls doesn't always mean contamination. Here's how to interpret NTC amplification and when it actually invalidates your data.
May 6, 2026How to Analyze qPCR Data with Biological Replicates
A practical guide to analyzing qPCR data with biological replicates — when to average, what to plot, and which statistical tests to run on ΔCt values.
May 5, 2026Absolute vs Relative Quantification in qPCR: Which One Do You Actually Need?
When to use absolute vs relative quantification in qPCR, how each method works, and the practical trade-offs most protocols gloss over.
May 4, 2026qPCR Standard Curve Best Practices and Common Mistakes
How to build a reliable qPCR standard curve: dilution series setup, efficiency calculations, and the mistakes that quietly wreck your absolute quantification.
May 1, 2026How to Normalize qPCR Data with Multiple Reference Genes
Using multiple reference genes in qPCR improves normalization accuracy. Here's how to combine them correctly using geNorm, NormFinder, or a simple geometric mean.
April 2, 2026Multiplex qPCR: A Practical Guide to Running Multiple Targets in One Well
How to design, optimize, and troubleshoot multiplex qPCR — primer/probe design, spectral crosstalk, efficiency matching, and common pitfalls.
March 23, 2026How to Design qPCR Primers That Actually Work
Practical primer design rules for qPCR: Tm matching, amplicon length, specificity checks, and the validation steps most protocols skip.
March 19, 2026The Pfaffl Method: When Your Primer Efficiencies Make ΔΔCt Unreliable
When qPCR primer efficiencies differ between target and reference genes, the Pfaffl method gives you accurate fold changes. Here's how to use it.
March 16, 2026Melt Curve Analysis in qPCR: What It Tells You and When to Worry
How to read melt curves in SYBR Green qPCR, spot primer dimers, identify off-target products, and decide when a run is trustworthy.
March 13, 2026SYBR Green vs TaqMan: When to Use Each Chemistry
A practical guide to choosing between SYBR Green and TaqMan for your qPCR experiments — based on cost, specificity, and what your project actually needs.
March 6, 2026Choosing the Best Reference Genes for qPCR
How to pick, validate, and use the right housekeeping genes for accurate normalization in RT-qPCR experiments.
March 6, 2026How to Calculate DDCt (Livak Method): A Complete Guide
Learn the delta-delta Ct method step by step, including the math, assumptions, common mistakes, and worked examples.
March 6, 2026qPCR Troubleshooting Guide: 10 Common Problems and Fixes
A practical checklist for diagnosing and fixing the most common qPCR issues, from NTC amplification to unexpected fold changes.