POTENTIAL RISKS PRESENT IN SELECTING CONTROL GENE FOR QUANTITATIVE RT-PCR: EXAMPLE OF MEASUREMENT USING 2D CELLS AND 3D SPHEROID OF ESOPHAGEAL CANCER CELLS

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Proper selection of the internal control gene is important for ensuring the accuracy of expression data obtained by quantitative real-time PCR. The expression of the internal control gene must remain constant under different experimental conditions. Typical validation methods include comparing the copy number per quantity of RNA or cDNA and normalizing multiple candidate internal control genes to each other. Both methods have advantages and disadvantages. Validation of internal control expression is essential for ensuring accurate experimental results. Even for frequently used internal controls such as β-actin and GAPDH, validation is necessary to ensure that no fluctuations in expression occur under different experimental conditions. In this report, we indicate concrete examples of the risks involved in normalizing candidate internal control genes to each other and how to complement this method. Internal control genes should be selected by establishing multiple combinations of candidate genes and comparing the copy number per quantity of RNA or cDNA.