Shi Leming, Tong Weida, Su Zhenqiang, Han Tao, Han Jing, Puri Raj K, Fang Hong, Frueh Felix W, Goodsaid Federico M, Guo Lei, Branham William S, Chen James J, Xu Z Alex, Harris Stephen C, Hong Huixiao, Xie Qian, Perkins Roger G, Fuscoe James C
National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, Arkansas 72079, USA.
BMC Bioinformatics. 2005 Jul 15;6 Suppl 2(Suppl 2):S11. doi: 10.1186/1471-2105-6-S2-S11.
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.
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 provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias.
It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.
基于微阵列的mRNA丰度测量假定荧光强度与染料浓度之间存在线性关系。然而，实际上校准曲线可能是非线性的。
通过在18种光电倍增管（PMT）增益条件下扫描包含已知浓度荧光染料的微阵列扫描仪校准载玻片，我们能够评估Cy5和Cy3校准特性的差异。首先，在相同PMT增益下，同一染料的校准曲线在高强度和低强度两端均呈非线性。其次，校准曲线的非线性程度取决于PMT增益。第三，即使在相同增益下，两个PMT（用于Cy5和Cy3）的表现也不同。第四，Cy3通道的背景强度高于Cy5通道。证明了这些特性对测量的mRNA丰度和计算比率的准确性及可重复性的影响。结合模拟结果，我们对染料交换重复实验中比率低估、比率偏差的强度依赖性以及比率的反相关性的存在给出了解释。我们进一步证明，尽管局部加权回归（Lowess）归一化有效地消除了比率偏差的强度依赖性，但与真实比率的系统偏差在很大程度上仍然存在。提出了一种基于从校准曲线估计的浓度来计算比率的方法，以校正比率偏差。
最好在固定的最佳增益设置下扫描微阵列载玻片，在该设置下浓度与强度之间的线性关系最大化。尽管归一化方法提高了微阵列测量的可重复性，但它们在提高准确性方面似乎效果较差。
