Basics of quantitative PCR 1: image analysis and quantitation of PCR products.

The reproducibility of quantitative image analytic assessment of agarose gel separated PCR products was tested in a computerised core facility image analysis unit serving several research groups. Routine type use of the instrumentation showed considerable variation (measurements of individuals bands: CV about 20%; ratio measurements: CV about 6.5%). Careful concentration on the methodology improved the result (CV about 9% and 4.5%, respectively). Focusing into the methodology clearly showed that the baseline correction was necessary. The magnification of the image and the width of the measurement window were important variation sources. If these were kept constant, the reproducibility improved remarkably (CVs of 1.69% and 2.90%, respectively). Further analysis showed that the results did not change during the warm up period of the instrumentation. Measurements based on integrated optical density or related variables were well correlated, but did not show linear relation with peak height estimates. The most reproducible results were achieved when the thresholds were placed at the point of 45 degrees inclination of the image density curve of the band, or at the half height points of the curve.