Article

Evaluation of five different cDNA labeling methods for microarrays using spike controls

BMC Biotechnology 01/2003;
Source: DOAJ

ABSTRACT 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 μg.

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.

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Keywords

48% respectively
 
A. thaliana
 
cDNA labeling methods
 
CyScribe labeling methods
 
different cDNA labeling methods
 
direct labeling
 
FairPlay method
 
FairPlay™ – aminoallyl
 
five different labeling methods
 
gene expression analysis
 
hapten-antibody enzymatic labeling
 
indirect labeling
 
largest deviation
 
largest experimental variation
 
lowest total experimental variation
 
microarray experiments
 
predetermined ratio values
 
relative deviation
 
spike controls
 
target spike mRNAs
 

Azadeh Badiee