Background: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors.
Methods: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe-amplicon hybrids were detected by a streptavidin-based method.
Results: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base.
Conclusions: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.
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