Differential display method used for the comparison of mRNA expression level among many samples

Abstract

A reproducibility of experimental data is important for application of differential display method to the comparison of mRNA expression level among many samples. First, we developed the optimum method to evaluate size (BP) and yield (IOD) of PCR products by analyzing an electrophoretic image. BP (A), which is a way to calculate size of each product by using 3 molecular markers loaded in extreme and central lanes of gel as references, was optimal for size evaluation. Further, IOD (A) and IOD (P), respectively, which are way to use a measured absolute value of signal intensity of each product, and to use a relative value of signal intensity of each product as proportion of total sum of all products in an identical lane, as yield, were determined as the suboptimum and optimum evaluation methods. Next, we performed an analysis of variance of intergel, interlane, and interreaction effects, when using the optimum method. We detected statistically significant effects for intergel and interlane, but not for interreaction, when using BP (A) method. As for IOD (A) and IOD (P), significant effects for intergel and interreaction were observed, but not for interlane. Finally, we evaluated the replicate pattern that is suitable for a largescale gene expression profiling, by examining coefficient of variation of combined effects for BP and IOD obtained by 4 different replicate patterns.