Fig. 1

Design and optimization of the qPCR assay. a Primer region selection for TYMS target gene used for expression analysis. NCBI Reference Sequence was NM_001071.2 (Homo sapiens thymidilate synthetase, mRNA). b Specificity for TYMS mRNA sequence was demonstrated in a 2 % agarose gel electrophoresis loaded with 10 μl PCR products from 3 random FFPE samples. TYMS products are expected to be 95 bp long. DNA Molecular Weight Marker XIII 50 bp ladder (Roche). c Primer efficiency for the TYMS qPCR assay. The efficiency of the primer pair was assessed by plotting the cycle threshold value (Cp) at each concentration against the logarithm of the fold dilution of the sample. The slope of a linear-regression trend line is indicative of primer efficiency. d Primer region selection for ATP5E reference gene. NCBI Reference Sequence was NM_006886.3 (Homo sapiens ATP synthase, H+ transporting, mitochondrial F1 complex, epsilon subunit, mRNA). e Specificity for ATP5E mRNA sequence was demonstrated in a 2 % agarose gel electrophoresis loaded with 10 μl PCR products from 3 random FFPE samples. ATP5E products are expected to be 101 bp long. f Primer efficiency for the ATP5E qPCR assay