Real Time PCR data analysis using Pfaffle method | step-by-step tutorial |

Описание: Real Time PCR data analysis using Pfaffle method: a method over ΔΔCT

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The delta Ct ( Δ 𝐶 𝑡 ΔCt) method is a widely used and straightforward approach for analyzing qPCR data to assess relative gene expression levels. This method involves normalizing the expression level of a target gene to that of a reference (or housekeeping) gene within the same sample. Housekeeping genes are chosen because they are assumed to have stable expression across different samples and experimental conditions. Overview of the Methodology Normalization to Reference Gene: The first step in the delta Ct method is to calculate the difference in Ct values ( Δ 𝐶 𝑡 ΔCt) between the target gene and the reference gene for each sample. This normalization step accounts for variations in the amount of input RNA and efficiency of reverse transcription. Comparison Across Conditions: If comparing gene expression between different groups (e.g., treated vs. control), further normalization is performed by comparing the Δ 𝐶 𝑡 ΔCt values of each sample to a reference condition, usually the untreated or control group. This is called the Δ Δ 𝐶 𝑡 ΔΔCt method. Relative Quantification: Finally, the fold change in gene expression is calculated using the formula 2 − Δ Δ 𝐶 𝑡 2 −ΔΔCt , providing a measure of relative expression levels.

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