Comparison of different labeling methods for two-channel high-density microarray experiments.
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 microg of total RNA compared with 20 microg 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.
Center for Bioinformatics, Univ. of Pennsylvania, Philadelphia, Pennsylvania 19104-6021, USA. manduchi@pcbi.upenn.edu
Elisabetta
E
Manduchi
L Marie
LM
Scearce
John E
JE
Brestelli
Gregory R
GR
Grant
Klaus H
KH
Kaestner
Christian J
CJ
Stoeckert
D005456:Fluorescent Dyes / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D012313:RNA