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Physiol. Genomics 10: 169-179, 2002. First published June 18, 2002; doi:10.1152/physiolgenomics.00120.2001
1094-8341/02 $5.00
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Received 18 December 2001; accepted in final form 12 June 2002.
Physiological Genomics 10:169-179 (2002)
1094-8341/02 $5.00 © 2002 American Physiological Society

Comparison of different labeling methods for two-channel high-density microarray experiments

Elisabetta Manduchi 1, L. Marie Scearce 2, John E. Brestelli 2, Gregory R. Grant 1, Klaus H. Kaestner 2 and Christian J. Stoeckert, JR. 1

1 Center for Bioinformatics, Philadelphia, Pennsylvania 19104
2 Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

In this report we evaluate three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere). The dendrimer method requires the smallest quantity of sample, 2.5 µg of total RNA compared with 20 µg with the direct or indirect methods. Therefore, we wanted to know whether the performance of the dendrimer method is comparable to the other methods, or whether significant information is lost. Performance can be considered in terms of sensitivity, dynamic range, and reproducibility of the quantitative signals for gene intensity. We compared the three labeling methods by generating three sets of eight self-to-self hybridizations using the same total RNA sample in all cases ("replicate study"). In our analysis, we controlled for the effects of print-tip and background subtraction biases. We also performed a smaller study, namely, a dilution series study with five dilution points per labeling method, to evaluate one aspect of predictive ability. From the replicate study, the dendrimer method appeared to perform as well, and often better, with respect to reproducibility and ability to detect expression. However, in the dilution series study, this method was outperformed by the other two in terms of predictive ability and did not perform very well. These findings are helping to guide our decisions on what labeling method to use for subsequent studies, based on the purpose of a specific study and its limitations in terms of available material.

evaluation of labeling protocols; direct labeling; indirect amino-allyl labeling; dendrimer labeling




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