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As negative controls, we included: (1) RNA extracted from Staq cell culture, known to be MAGE-A3-negative; (2) water submitted to reverse transcriptase and PCR steps and (3) only water submitted to PCR steps.
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Briefly, kDNA amplicons were subjected to electrophoresis in 1.5% agarose gel (1.0% agarose, 0.5% agarose low melting point), punctured from the gel, diluted 10 times in double distilled water, and submitted to a second step of low stringency amplification, using a single primer (S35G: 5'-ATGTACGGGGAGATGCATGA-3').
The aqueous layer was isolated, lyophilized, resuspended in water, and submitted for LC-MS/MS analysis for PTC-ACC.
Normalized cDNA in 100 µl sterile water was submitted to the Georgia Genomic Facility (www.dna.uga.edu) for 454 sequencing in a Genome Sequencer TM (GS) Titanium FLX instrument (Roche Diagnostics) employing a standard protocol.
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