IDH mutation analysis in bone marrow cells from myeloid neoplasms. The figure illustrates the large number of samples analyzed (black columns) but absence of IDH mutated cases in this cohort of Ph⁻ MPN and MDS. DNA was extracted with the DNeasy kit (Qiagen). The following custom-made primer sets were used for mutant allele quantification at IDH1 codon R132 (forward 5′-biotin-gcttgtgagtggatgggtaaa, reverse 5′-gacttacttgatccccataagca, 65 bp; sequencing primer-1, 5′-tccccataagcatgac, and primer-2, 5′-gatccccataagcatg, covering the hotspot bases in codon R132) and IDH2 codon R172 (forward 5′-atcccacgcctagtccct, reverse 5′-biotin-tctccaccctggcctacc, 83 bp; sequencing primer 5′-ccatcaccattggca). Wild-type status of one control was confirmed by capillary sequencing: IDH1 (forward 5′-caagttgaaacaaatgtggaaatcac, reverse 5′-gtcacttggtgtgtaggttatctctctac, 223 bp) and IDH2 (forward 5′-tggaagagttcaagctgaagaagat, reverse 5′-tgccatcttttggggtgaag, 237 bp).