Abstract
Background
Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay--aminoallyl), two protocols with dendrimer technology (3DNA Array 50 and 3DNA submicro), and hapten-antibody enzymatic labeling (Micromax TSA). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5-50 microg.Results
The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively).Conclusion
We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.