The detection of MAGE-A3 RNA expression by reverse transcription PCR Routine protocols and reagents were used for the extraction of total RNA (with DNAse digestion step) from manually dissected formalin-fixed paraffin-embedded tumor sections (containing at least 80% tumor cells) and from the human melanoma cell line MZ-2-3.0 (positive control; provided by Ludwig Institute of Cancer Research, Brussels, Belgium); and routine protocols and reagents were used for the preparation of cDNA from this RNA. and (control) cDNA fragments were amplified by PCR using TaqMan MAGE-A3 ACTB reagents (Thermo Fisher Scientific, Gent, Belgium), the ABI 7900 system (Thermo Fisher Scientific, Gent, Belgium), and the following primers; probe MAGE-A3 forward TGT-CGT-CGG-AAA-TTG-GCA-GTA-T MAGE-A3 and reverse CAA-AGA-CCA-GCT-GCA-AGG-AAC-T, probe MAGE-A3 ACTB forward CTG-GAA-CGG-TGA-AGG-TGA-CA, and reverse ACTB ACTB CGG-CCA-CAT-TGT-GAA-CTT-TG. The multi-well plates used in the PCR contained cDNA from tumor specimens and the positive control. A tumor specimen was defined as positive if the relative expression of to in the specimen was MAGE-A3 MAGE-A3 ACTB ≥1% of the relative expression of to in the positive control. MAGE-A3 ACTB Study design details The criteria for adequate bone-marrow reserve were; white blood-cell count > 3 x 109 /l, neutrophils > 1.5 109/l, platelets > 50 109/l, lymphocytes > 0.5 109/l, and hemoglobin > 10.0 g/dl. The criterion for adequate renal function was creatinine < 3-fold the upper limit of normal and the criteria for adequate hepatic function were serum bilirubin within normal range, and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase all <2-fold the upper limit of normal.
The detection of MAGE-A3 RNA expression by reverse transcription PCR Routine protocols and reagents were used for the extraction of total RNA (with DNAse digestion step) from manually dissected formalin-fixed paraffin-embedded tumor sections (containing at least 80% tumor cells) and from the human melanoma cell line MZ-2-3.0 (positive control; provided by Ludwig Institute of Cancer Research, Brussels, Belgium); and routine protocols and reagents were used for the preparation of cDNA from this RNA. MAGE-A3 and ACTB (control) cDNA fragments were amplified by PCR using TaqMan reagents (Thermo Fisher Scientific, Gent, Belgium), the ABI 7900 system (Thermo Fisher Scientific, Gent, Belgium), and the following primers; MAGE-A3 probe MAGE-A3 forward TGT-CGT-CGG-AAA-TTG-GCA-GTA-T and MAGE-A3 reverse CAA-AGA-CCA-GCT-GCA-AGG-AAC-T, ACTB probe ACTB forward CTG-GAA-CGG-TGA-AGG-TGA-CA, and ACTB reverse CGG-CCA-CAT-TGT-GAA-CTT-TG. The multi-well plates used in the PCR contained cDNA from tumor specimens and the positive control. A tumor specimen was defined as MAGE-A3 positive if the relative expression of MAGE-A3 to ACTB in the specimen was ≥1% of the relative expression of MAGE-A3 to ACTB in the positive control. Study design details The criteria for adequate bone-marrow reserve were; white blood-cell count > 3 x 109 /l, neutrophils > 1.5 109/l, platelets > 50 109/l, lymphocytes > 0.5 109/l, and hemoglobin > 10.0 g/dl. The criterion for adequate renal function was creatinine < 3-fold the upper limit of normal and the criteria for adequate hepatic function were serum bilirubin within normal range, and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase all <2-fold the upper limit of normal.