Microarray scanner calibration curves: characteristics and implications

Background: Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear. Results: By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we pr...

• Type: Journal
• Year: 2005
• Where: BMCBI
• Authors: Leming M. Shi, Weida Tong, Zhenqiang Su, Tao Han, Jing Han, Raj K. Puri, Hong Fang, Felix W. Frueh, Federico M. Goodsaid, Lei Guo, William S. Branham, James J. Chen, Z. Alex Xu, Stephen C. Harris, Huixiao Hong, Qian Xie, Roger Perkins, James C. Fuscoe
• Keywords: BMCBI 2005, Calibration Curve, Pmt Gain, Ratio Bias