Hello,
I prepare PCR tests to check for stemness markers. I compare cells from 2D and 3D cultures, as well as tumor tissue. Although I can normalize 2D and tumor tissue using RNA isolation and measuring RNA concentration(Total RNA Mini - AABiotech) , I use the TaqMan™ Gene Expression Cells-to-CT™ Kit for 3D RNA isolation where RNA concentration is not measured. Is there any way to compare qPCR results in this way? Has anyone experienced with problem like that? Thanks in advance for any suggestions.
Comparing qPCR results requires systematic analysis of amplification curves, Cq values, and expression levels while controlling for variability. Begin by examining amplification curves for proper sigmoidal shapes and exclude any outliers or failed replicates with late Cq values (>35) or irregular profiles. Ensure consistent baseline and threshold settings across all runs for comparable Cq measurements. For meaningful comparison, normalize target gene Cq values against stable reference genes (like GAPDH or β-actin) using the ΔCq method (ΔCq = Cq_target - Cq_reference), first verifying reference gene stability with tools like geNorm. When comparing experimental groups, use the ΔΔCq method (ΔΔCq = ΔCq_test - ΔCq_control) to calculate fold-change expression (2^-ΔΔCq). Always average technical replicates while removing outliers with high variability, then analyze biological replicates using appropriate statistics (t-tests or ANOVA). For absolute quantification, create a standard curve with known DNA concentrations. If PCR efficiency isn't ideal (90-110%), incorporate efficiency corrections into fold-change calculations. Visualize results using bar plots (mean ± SD) or heatmaps, and consider using specialized software like qBase+ or GraphPad Prism. Key pitfalls to avoid include ignoring PCR efficiency differences, contamination in no-template controls, and inter-run variability - always run samples in the same plate when possible. For more precise analysis, efficiency-corrected models can replace the classic 2^-ΔΔCq method when reaction efficiencies deviate significantly from 100%.
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