DNA microarray to monitor the expression of MAGE-A genes.
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.
Zammatteo
Nathalie
N
Laboratoire de Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium. nathalie.zammatteo@fundp.ac.be
Lockman
Laurence
L
Brasseur
Francis
F
De Plaen
Etienne
E
Lurquin
Christophe
C
Lobert
Pierre-Emmanuel
PE
Hamels
Sandrine
S
Boon
Thierry
T
Remacle
José
J
D000951:Antigens, Neoplasm / Q000235:genetics*; D006801:Humans; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; C119317:MAGE-A10 antigen; C409589:MAGEA12 protein, human; C072013:MAGEA3 protein, human; C095204:MAGEA4 protein, human; C420909:Mage-a2 antigen; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins