Unambiguous Identification of the Expressed MAGE-A Genes on a DNA Microarray

We have described a post-PCR detection method for the 12 MAGE-A sequences on a DNA microarray (1) and compared the results with a method using pairs of primers unique for each sequence (2). The microarray assay did not differentiate between MAGE-A3 and MAGE-A6 amplicons, which differed by only 1 nucleotide, and could not distinguish between PCR products amplified from mRNA and those amplified from genomic DNA. In addition, the assay could not identify false-negative results related to RNA degradation or to enzyme inhibition during reverse transcription-PCR. Here we describe an assay that is designed to overcome these drawbacks and appears to be more sensitive. We used 3 primer pairs: 1 for amplification of the 12 MAGE-A sequences (1); 1 for specific amplification of MAGE-A3 ; and 1 for amplification of an endogenous, ubiquitously expressed control gene ( MAGE-D2 ) (3). The low-density microarray includes new capture probes for MAGE-A3 and - D2 . The assay involved DNase treatment of total RNA, reverse transcription of mRNA with oligo(dT) primer, triplex PCR amplification in the presence of biotin-dATP/biotin-dCTP, and hybridization of the resulting amplicons on a DNA microarray (see the Experimental Protocol in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol51/issue12). The new capture probe for MAGE-A3 corresponded to a sequence downstream from the location of the reverse primer DPASCONB4, and a primer pair (DPSA3 and DPASA3) defining a MAGE-A3 amplicon encompassing the new probe sequence …